Separation Matrix

ABSTRACT

The invention relates to a separation matrix comprising at least 11 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
     a) the ligands comprise multimers of alkali-stabilized Protein A domains, and   b) the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 17/221,438,filed Apr. 2, 2021, which is a continuation of U.S. Application No.16,893,574, filed Jun. 5, 2020, now U.S. Pat. No. 10,995,113, issuedJun. 4, 2021, which is a continuation of U.S. Applciation No.16/682,855, filed Nov. 13, 2019, now U.S. Pat. No. 10,711,035, issuedJul. 14, 2020, which is a continuation of U.S. Application No.16/096,952, filed Oct. 26, 2018, now U.S. Pat. No. 10,513,537, issuedDec. 24, 2019, which claims the priority benefit of PCT/EP2017/061164filed on May 10, 2017, which claims priority benefit of U.S. ApplicationNo. 15/282,367, filed Sep. 30, 2016, and Great Britain Application Nos.1608229.9 and 1608232.3, both of which were filed May 11, 2016. Theentire contents of which are hereby incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 22, 2018, isnamed 313838A_ST26.XML and is 104,000 bytes in size.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of affinity chromatography,and more specifically to mutated immunoglobulin-binding domains ofProtein A, which are useful in affinity chromatography ofimmunoglobulins. The invention also relates to multimers of the mutateddomains and to separation matrices containing the mutated domains ormultimers.

BACKGROUND OF THE INVENTION

Immunoglobulins represent the most prevalent biopharmaceutical productsin either manufacture or development worldwide. The high commercialdemand for and hence value of this particular therapeutic market has ledto the emphasis being placed on pharmaceutical companies to maximize theproductivity of their respective mAb manufacturing processes whilstcontrolling the associated costs.

Affinity chromatography is used in most cases, as one of the key stepsin the purification of these immunoglobulin molecules, such asmonoclonal or polyclonal antibodies. A particularly interesting class ofaffinity reagents is proteins capable of specific binding to invariableparts of an immunoglobulin molecule, such interaction being independenton the antigen-binding specificity of the antibody. Such reagents can bewidely used for affinity chromatography recovery of immunoglobulins fromdifferent samples such as but not limited to serum or plasmapreparations or cell culture derived feed stocks. An example of such aprotein is staphylococcal protein A, containing domains capable ofbinding to the Fc and Fab portions of IgG immunoglobulins from differentspecies. These domains are commonly denoted as the E-, D-, A-, B- andC-domains.

Staphylococcal protein A (SpA) based reagents have due to their highaffinity and selectivity found a widespread use in the field ofbiotechnology, e.g. in affinity chromatography for capture andpurification of antibodies as well as for detection or quantification.At present, SpA-based affinity medium probably is the most widely usedaffinity medium for isolation of monoclonal antibodies and theirfragments from different samples including industrial cell culturesupernatants. Accordingly, various matrices comprising protein A-ligandsare commercially available, for example, in the form of native protein A(e.g. Protein A SEPHAROSE, GE Healthcare, Uppsala, Sweden) and alsocomprised of recombinant protein A (e.g. rProtein A-SEPHAROSE, GEHealthcare). More specifically, the genetic manipulation performed inthe commercial recombinant protein A product is aimed at facilitatingthe attachment thereof to a support and at increasing the productivityof the ligand.

These applications, like other affinity chromatography applications,require comprehensive attention to definite removal of contaminants.Such contaminants can for example be non-eluted molecules adsorbed tothe stationary phase or matrix in a chromatographic procedure, such asnon-desired biomolecules or microorganisms, including for exampleproteins, carbohydrates, lipids, bacteria and viruses. The removal ofsuch contaminants from the matrix is usually performed after a firstelution of the desired product, in order to regenerate the matrix beforesubsequent use. Such removal usually involves a procedure known ascleaning-in-place (CIP), wherein agents capable of eluting contaminantsfrom the stationary phase are used. One such class of agents often usedis alkaline solutions that are passed over said stationary phase. Atpresent the most extensively used cleaning and sanitizing agent is NaOH,and the concentration thereof can range from 0.1 up to e.g. 1 M,depending on the degree and nature of contamination. This strategy isassociated with exposing the matrix to solutions with pH-values above13. For many affinity chromatography matrices containing proteinaceousaffinity ligands such alkaline environment is a very harsh condition andconsequently results in decreased capacities owing to instability of theligand to the high pH involved.

An extensive research has therefore been focused on the development ofengineered protein ligands that exhibit an improved capacity towithstand alkaline pH-values. For example, Gülich et al. (SusanneGülich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober,Journal of Biotechnology 80 (2000), 169-178) suggested proteinengineering to improve the stability properties of a Streptococcalalbumin-binding domain (ABD) in alkaline environments. Gülich et al.created a mutant of ABD, wherein all the four asparagine residues havebeen replaced by leucine (one residue), aspartate (two residues) andlysine (one residue). Further, Gülich et al. report that their mutantexhibits a target protein binding behavior similar to that of the nativeprotein, and that affinity columns containing the engineered ligand showhigher binding capacities after repeated exposure to alkaline conditionsthan columns prepared using the parental non-engineered ligand. Thus, itis concluded therein that all four asparagine residues can be replacedwithout any significant effect on structure and function.

Recent work shows that changes can also be made to protein A (SpA) toeffect similar properties. U.S. Pat. application publication US2005/0143566, which is hereby incorporated by reference in its entirety,discloses that when at least one asparagine residue is mutated to anamino acid other than glutamine or aspartic acid, the mutation confersan increased chemical stability at pH-values of up to about 13-14compared to the parental SpA, such as the B-domain of SpA, or Protein Z,a synthetic construct derived from the B-domain of SpA (US 5,143,844,incorporated by reference in its entirety). The authors show that whenthese mutated proteins are used as affinity ligands, the separationmedia as expected can better withstand cleaning procedures usingalkaline agents. Further mutations of protein A domains with the purposeof increasing the alkali stability have also been published in US8,329,860, JP 2006304633A, US 8,674,073, US 2010/0221844, US2012/0208234, US 9,051,375, US 2014/0031522, US 2013/0274451 and WO2014/146350, all of which are hereby incorporated by reference in theirentireties. However, the currently available mutants are still sensitiveto alkaline pH and the NaOH concentration during cleaning is usuallylimited to 0.1 M, which means that complete cleaning is difficult toachieve. Higher NaOH concentrations, which would improve the cleaning,lead to unacceptable capacity losses.

There is thus still a need in this field to obtain a separation matrixcontaining protein ligands having a further improved stability towardsalkaline cleaning procedures. There is also a need for such separationmatrices with an improved binding capacity to allow for economicallyefficient purification of therapeutic antibodies.

SUMMARY OF THE INVENTION

One aspect of the invention is to provide a polypeptide with improvedalkaline stability. This is achieved with an Fc-binding polypeptidecomprising a mutant of a parental Fc-binding domain of StaphylococcusProtein A (SpA), as defined by, or having at least 80% such as at least90%, 95% or 98% identity to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQID NO:51 or SEQ ID NO:52, wherein at least the asparagine or serineresidue at the position corresponding to position 11 in SEQ ID NO:4-7has been mutated to an amino acid selected from the group consisting ofglutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine,isoleucine, tryptophan, methionine, valine, alanine, histidine andarginine. Alternatively, the polypeptide comprises a sequence as definedby, or having at least 80% or at least 90%, 95% or 98% identity to SEQID NO:53.

X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉ SX₁₀X₁₁X₁₂LAEAKX₁₃X₁₄NX₁₅AQ (SEQ ID NO:53)

wherein individually of each other:

-   X₁=A or Q or is deleted-   X₂=E,K,Y,T,F,L,W,I,M,V,A,H or R-   X₃=H or K-   X₄=A or N-   X₅=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K-   X₆=Q or E-   X₇=S or K-   X₈=E or D-   X₉=Q or V or is deleted-   X₁₀=K,R or A or is deleted-   X₁₁=A,E or N or is deleted-   X₁₂=I or L-   X₁₃=K or R-   X₁₄=L or Y-   X₁₅=D, F,Y,W,K or R

One advantage is that the alkaline stability is improved over theparental polypeptides, with a maintained highly selective bindingtowards immunoglobulins and other Fc-containing proteins.

A second aspect of the invention is to provide a multimer with improvedalkaline stability, comprising a plurality of polypeptides. This isachieved with a multimer of the polypeptide disclosed above.

A third aspect of the invention is to provide a nucleic acid or a vectorencoding a polypeptide or multimer with improved alkaline stability.This is achieved with a nucleic acid or vector encoding a polypeptide ormultimer as disclosed above.

A fourth aspect of the invention is to provide an expression systemcapable of expressing a polypeptide or multimer with improved alkalinestability. This is achieved with an expression system comprising anucleic acid or vector as disclosed above.

A fifth aspect of the invention is to provide a separation matrixcapable of selectively binding immunoglobulins and other Fc-containingproteins and exhibiting an improved alkaline stability. This is achievedwith a separation matrix comprising at least 11 mg/ml Fc-binding ligandscovalently coupled to a porous support, wherein:

-   a) the ligands comprise multimers of alkali-stabilized Protein A    domains,-   b) the porous support comprises cross-linked polymer particles    having a volume-weighted median diameter (d50,v) of 56-70    micrometers and a dry solids weight of 55-80 mg/ml. Alternatively,    it is achieved with a separation matrix comprising at least 15 mg/ml    Fc-binding ligands covalently coupled to a porous support, wherein    said ligands comprise multimers of alkali-stabilized Protein A    domains.

One advantage is that a high dynamic binding capacity is provided. Afurther advantage is that a high degree of alkali stability is achieved.

A sixth aspect of the invention is to provide an efficient andeconomical method of isolating an immunoglobulin or other Fc-containingprotein. This is achieved with a method comprising the steps of:

-   a) contacting a liquid sample comprising an immunoglobulin with a    separation matrix as disclosed above,-   b) washing the separation matrix with a washing liquid,-   c) eluting the immunoglobulin from the separation matrix with an    elution liquid, and-   d) cleaning the separation matrix with a cleaning liquid.

Further suitable embodiments of the invention are described in thedependent claims. Copending applications PCT EP2015/076639, PCTEP2015/076642, GB 1608229.9 and GB 1608232.3 are hereby incorporated byreference in their entireties.

DEFINITIONS

The terms “antibody” and “immunoglobulin” are used interchangeablyherein, and are understood to include also fragments of antibodies,fusion proteins comprising antibodies or antibody fragments andconjugates comprising antibodies or antibody fragments.

The terms an “Fc-binding polypeptide” and “Fc-binding protein” mean apolypeptide or protein respectively, capable of binding to thecrystallisable part (Fc) of an antibody and includes e.g. Protein A andProtein G, or any fragment or fusion protein thereof that has maintainedsaid binding property.

The term “linker” herein means an element linking two polypeptide units,monomers or domains to each other in a multimer.

The term “spacer” herein means an element connecting a polypeptide or apolypeptide multimer to a support.

The term “% identity” with respect to comparisons of amino acidsequences is determined by standard alignment algorithms such as, forexample, Basic Local Alignment Tool (BLAST) described in Altshul et al.(1990) J. Mol. Biol., 215: 403-410. A web-based software for this isfreely available from the US National Library of Medicine atblast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINKLOC =blasthome. Here, the algorithm “blastp (protein-protein BLAST)” isused for alignment of a query sequence with a subject sequence anddetermining i.a. the % identity.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S. Pat.law and can mean “includes,” “including,” and the like; “consistingessentially of or “consists essentially” likewise has the meaningascribed in U.S. Pat. law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an alignment of the Fc-binding domains as defined by SEQ IDNO: 1-7 and 51-52.

FIG. 2 shows results from Example 2 for the alkali stability of parentaland mutated tetrameric Zvar (SEQ ID NO:7) polypeptide variants coupledto an SPR biosensor chip.

FIG. 3 shows results from Example 4 for the alkali stability (0.5 MNaOH) of parental and mutated tetrameric Zvar (SEQ ID NO:7) polypeptidevariants coupled to agarose beads.

FIG. 4 shows results from Example 4 for the alkali stability (1.0 MNaOH) of parental and mutated tetrameric Zvar (SEQ ID NO:7) polypeptidevariants coupled to agarose beads.

FIG. 5 shows results from Example 7 for the alkali stability (1.0 MNaOH) of agarose beads with different amounts of mutated multimervariants (SEQ ID NO:20) coupled. The results are plotted as the relativeremaining dynamic capacity (Qb10%, 6 min residence time) vs. incubationtime in 1 M NaOH.

FIG. 6 shows results from Example 7 for the alkali stability (1.0 MNaOH) of agarose beads with different amounts of mutated multimervariants (SEQ ID NO:20) coupled. The results are plotted as the relativeremaining dynamic capacity (Qb10%, 6 min residence time) after 31 hincubation in 1 M NaOH vs. the ligand content of the prototypes.

DETAILED DESCRIPTION OF EMBODIMENTS

In one aspect the present invention discloses an Fc-binding polypeptide,which comprises, or consists essentially of, a mutant of an Fc-bindingdomain of Staphylococcus Protein A (SpA), as defined by, or having atleast 90%, at least 95% or at least 98% identity to, SEQ ID NO:1(E-domain), SEQ ID NO:2 (D-domain), SEQ ID NO:3 (A-domain), SEQ ID NO:22(variant A-domain), SEQ ID NO:4 (B-domain), SEQ ID NO:5 (C-domain), SEQID NO:6 (Protein Z), SEQ ID NO:7 (Zvar), SEQ ID NO:51 (Zvar without thelinker region amino acids 1-8 and 56-58) or SEQ ID NO:52 (C-domainwithout the linker region amino acids 1-8 and 56-58) as illustrated inFIG. 1 , wherein at least the asparagine (or serine, in the case of SEQID NO:2) residue at the position* corresponding to position 11 in SEQ IDNO:4-7 has been mutated to an amino acid selected from the groupconsisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine,leucine, isoleucine, tryptophan, methionine, valine, alanine, histidineand arginine. Protein Z (SEQ ID NO:6) is a mutated B-domain as disclosedin US5143844, hereby incorporated by reference in its entirety, whileSEQ ID NO:7 denotes a further mutated variant of Protein Z, here calledZvar, with the mutations N3A,N6D,N23T. SEQ ID NO:22 is a natural variantof the A-domain in Protein A from Staphylococcus aureus strain N315,having an A46S mutation, using the position terminology of FIG. 1 . Themutation of N11 in these domains confers an improved alkali stability incomparison with the parental domain/polypeptide, without impairing theimmunoglobulin-binding properties. Hence, the polypeptide can also bedescribed as an Fc- or immunoglobulin-binding polypeptide, oralternatively as an Fc- or immunoglobulin-binding polypeptide unit.

*Throughout this description, the amino acid residue position numberingconvention of FIG. 1 is used, and the position numbers are designated ascorresponding to those in SEQ ID NO:4-7. This applies also to multimers,where the position numbers designate the positions in the polypeptideunits or monomers according to the convention of FIG. 1 .

SEQ ID NO:51 (truncated Zvar)

QQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQ

SEQ ID NO:52 (truncated C domain)

QQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKEILAEAKK LNDAQ

In alternative language, the invention discloses an Fc-bindingpolypeptide which comprises a sequence as defined by, or having at least90%, at least 95% or at least 98% identity to SEQ ID NO:53.

SEQ ID NO:53

X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉ SX₁₀X₁₁X₁₂LAEAKX₁₃X₁₄NX₁₅AQ

wherein individually of each other:

-   X₁=A, Q or is deleted-   X₂=E,K,Y,T,F,L,W,I,M,V,A,H or R-   X₃=H or K-   X₄=A or N-   X₅=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, such as    S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K-   X₆=Q or E-   X₇=S or K-   X₈=E or D-   X₉=Q, V or is deleted-   X₁₀=K, R, A or is deleted-   X₁₁=A, E, N or is deleted-   X₁₂=I or L-   X₁₃=K or R-   X₁₄=L or Y-   X₁₅=D, F,Y,W,K or R

Specifically, the amino acid residues in SEQ ID NO:53 may individuallyof each other be:

-   X₁=A or is deleted-   X₂=E-   X₃=H-   X₄=N-   X₆=Q-   X₇=S-   X₈=D-   X₉=V or is deleted-   X₁₀=K or is deleted-   X₁₁=A or is deleted-   X₁₂=I-   X₁₃=K-   X₁₄=L.

In certain embodiments, the amino acid residues in SEQ ID NO:53 may be:X₁=A, X₂ = E, X₃ = H, X₄ = N, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀ = K,X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = L. In some embodiments X₂ = E, X₃ = H,X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₁₂ = I, X₁₃ = K, X₁₄ = L andX₁₅=D and one or more of X₁, X₉, X₁₀ and X₁₁ is deleted. In furtherembodiments, X₁=A, X₂ = E, X₃ = H, X₄ = N, X₅=S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀= K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D, or alternativelyX₁=A, X₂ = E, X₃ = H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀= K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = Land X₁₅= F,Y,W,K or R.

The N11 (X₂) mutation (e.g. a N11E or N11K mutation) may be the onlymutation or the polypeptide may also comprise further mutations, such assubstitutions in at least one of the positions corresponding topositions 3, 6, 9, 10, 15, 18, 23, 28, 29, 32, 33, 36, 37, 40, 42, 43,44, 47, 50, 51, 55 and 57 in SEQ ID NO:4-7. In one or more of thesepositions, the original amino acid residue may e.g. be substituted withan amino acid which is not asparagine, proline or cysteine. The originalamino acid residue may e.g. be substituted with an alanine, a valine, athreonine, a serine, a lysine, a glutamic acid or an aspartic acid.Further, one or more amino acid residues may be deleted, e.g. frompositions 1-6 and/or from positions 56-58.

In some embodiments, the amino acid residue at the positioncorresponding to position 9 in SEQ ID NO:4-7 (X₁) is an amino acid otherthan glutamine, asparagine, proline or cysteine, such as an alanine orit can be deleted. The combination of the mutations at positions 9 and11 provides particularly good alkali stability, as shown by theexamples. In specific embodiments, in SEQ ID NO: 7 the amino acidresidue at position 9 is an alanine and the amino acid residue atposition 11 is a lysine or glutamic acid, such as a lysine. Mutations atposition 9 are also discussed in copending applicationPCT/SE2014/050872, which is hereby incorporated by reference in itsentirety.

In some embodiments, the amino acid residue at the positioncorresponding to position 50 in SEQ ID NO:4-7 (X₁₃) is an arginine or aglutamic acid.

In certain embodiments, the amino acid residue at the positioncorresponding to position 3 in SEQ ID NO:4-7 is an alanine and/or theamino acid residue at the position corresponding to position 6 in SEQ IDNO:4-7 is an aspartic acid. One of the amino acid residues at positions3 and 6 may be an asparagine and in an alternative embodiment both aminoacid residues at positions 3 and 6 may be asparagines.

In some embodiments the amino acid residue at the position correspondingto position 43 in SEQ ID NO:4-7 (X₁₁ is an alanine or a glutamic acid,such as an alanine or it can be deleted. In specific embodiments, theamino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanineand lysine/glutamic acid respectively, while the amino acid residue atposition 43 is alanine or glutamic acid.

In certain embodiments the amino acid residue at the positioncorresponding to position 28 in SEQ ID NO:4-7 (X₅) is an alanine or anasparagine, such as an alanine.

In some embodiments the amino acid residue at the position correspondingto position 40 in SEQ ID NO:4-7 (X₉) is selected from the groupconsisting of asparagine, alanine, glutamic acid and valine, or from thegroup consisting of glutamic acid and valine or it can be deleted. Inspecific embodiments, the amino acid residues at positions 9 and 11 inSEQ ID NO: 7 are alanine and glutamic acid respectively, while the aminoacid residue at position 40 is valine. Optionally, the amino acidresidue at position 43 may then be alanine or glutamic acid.

In certain embodiments, the amino acid residue at the positioncorresponding to position 42 in SEQ ID NO:4-7 (X₁₀) is an alanine,lysine or arginine or it can be deleted.

In some embodiments the amino acid residue at the position correspondingto position 18 in SEQ ID NO:4-7 (X₃) is a lysine or a histidine, such asa lysine.

In certain embodiments the amino acid residue at the positioncorresponding to position 33 in SEQ ID NO:4-7 (X₇) is a lysine or aserine, such as a lysine.

In some embodiments the amino acid residue at the position correspondingto position 37 in SEQ ID NO:4-7 (X₈) is a glutamic acid or an asparticacid, such as a glutamic acid.

In certain embodiments the amino acid residue at the positioncorresponding to position 51 in SEQ ID NO:4-7 (X₁₄) is a tyrosine or aleucine, such as a tyrosine.

In some embodiments, the amino acid residue at the positioncorresponding to position 44 in SEQ ID NO:4-7 (X₁₂) is a leucine or anisoleucine. In specific embodiments, the amino acid residues atpositions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acidrespectively, while the amino acid residue at position 44 is isoleucine.Optionally, the amino acid residue at position 43 may then be alanine orglutamic acid.

In some embodiments, the amino acid residues at the positionscorresponding to positions 1, 2, 3 and 4 or to positions 3, 4, 5 and 6in SEQ ID NO: 4-7 have been deleted. In specific variants of theseembodiments, the parental polypeptide is the C domain of Protein A (SEQID NO: 5). The effects of these deletions on the native C domain aredescribed in US9018305 and US8329860, which are hereby incorporated byreference in their entireties.

In certain embodiments, the mutation in SEQ ID NO:4-7, such as in SEQ IDNO:7, is selected from the group consisting of:N11K; N11E; N11Y; N11T;N11F; N11L; N11W; N11I; N11M; N11V; N11A; N11H; N11R; N11E,Q32A;N11E,Q32E,Q40E; N11E,Q32E,K50R; Q9A,N11E,N43A; Q9A,N11E,N28A,N43A;Q9A,N11E,Q40V,A42K,N43E,L44I; Q9A,N11E,Q40V,A42K,N43A,L44I;N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y;Q9A,N11E,N28A,Q40V,A42K,N43A,L44I;Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y; N11K, H18K, D37E,A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K,H18K, D37E, A42R, N43A, L44I, K50R; Q9A,N11K,H18K,D37E,A42R;Q9A,N11E,D37E,Q40V,A42K,N43A,L44I and Q9A,N11E,D37E,Q40V,A42R,N43A,L44I.These mutations provide particularly high alkaline stabilities. Themutation in SEQ ID NO:4-7, such as in SEQ ID NO:7, can also be selectedfrom the group consisting of N11K; N11Y; N11F; N11L; N11W; N11I; N11M;N11V; N11A; N11H; N11R; Q9A,N11E,N43A; Q9A,N11E,N28A,N43A;Q9A,N11E,Q40V,A42K,N43E,L44I; Q9A,N11E,Q40V,A42K,N43A,L44I;Q9A,N11E,N28A,Q40V,A42K,N43A,L44I;N11K,H18K,S33K,D37E,A42R,N43A,L441,K50R,L5 1Y;Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y; N11K, H18K, D37E,A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I and Q9A, N11K,H18K, D37E, A42R, N43A, L44I, K50R.

In some embodiments, the polypeptide comprises or consists essentiallyof a sequence defined by or having at least 90%, 95% or 98% identity toan amino acid sequence selected from the group consisting of: SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ IDNO:50. It may e.g. comprise or consist essentially of a sequence definedby or having at least 90%, 95% or 98% identity to an amino acid sequenceselected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29. It canalso comprise or consist essentially of a sequence defined by or havingat least 90%, 95% or 98% identity to an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40; SEQ ID NO:41;SEQ ID NO:42; SEQ NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQID NO:47 and SEQ ID NO:48.

In certain embodiments, the polypeptide comprises or consistsessentially of a sequence defined by or having at least 90%, 95% or 98%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO:54-70. comprises or consists essentially of a sequence definedby or having at least 90%, 95% or 98% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:71-75, or it maycomprise or consist essentially of a sequence defined by or having atleast 90%, 95% or 98% identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO:76-79. It may further comprise orconsist essentially of a sequence defined by or having at least 90%, 95%or 98% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:89-95.

The polypeptide may e.g. be defined by a sequence selected from thegroups above or from subsets of these groups, but it may also compriseadditional amino acid residues at the N-and/or C-terminal end, e.g. aleader sequence at the N-terminal end and/or a tail sequence at theC-terminal end.

SEQ ID NO:8 Zvar(Q9A,N11E,N43A)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK

SEQ ID NO:9 Zvar(Q9A,N11E,N28A,N43A)

VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK

SEQ ID NO:10 Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK

SEQ ID NO:11 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:12 Zvar(N11E,Q32A)

VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IASLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:13 Zvar(N11E)

VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:14 Zvar(N11E,Q32E,Q40E)

VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSE SANLLAEAKK LNDAQAPK

SEQ ID NO:15 Zvar(N11E,Q32E,K50R)

VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSQ SANLLAEAKR LNDAQAPK

SEQ ID NO:16 Zvar(N11K)

VDAKFDKEQQ KAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:23 Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)

VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

SEQ ID NO:24 Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:25 Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)

VDAKFDKEAQ KAFYEILKLP NLTEEQRAAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

SEQ ID NO:26 Zvar(N11K, H18K, D37E, A42R, N43A, L44I)

VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKK LNDAQAPK

SEQ ID NO:27 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)

VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKK LNDAQAPK

SEQ ID NO:28 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)

VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKR LNDAQAPK

SEQ ID NO:29 Zvar(Q9A,N11K,H18K,D37E,A42R)

VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPK

SEQ ID NO:36 B(Q9A,N11E,Q40V,A42K,N43A,L44I)

ADNKFNKEAQ EAFYEILHLP NLNEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:37 C(Q9A,N11E,E43A)

ADNKFNKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:38 Zvar(N11Y)

VDAKFDKEQQ YAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:39 Zvar(N11T)

VDAKFDKEQQ TAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:40 Zvar(N11F)

VDAKFDKEQQ FAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:41 Zvar(N11L)

VDAKFDKEQQ LAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:42 Zvar(N11W)

VDAKFDKEQQ WAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:43 Zvar(N11I)

VDAKFDKEQQ IAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:44 Zvar(N11M)

VDAKFDKEQQ MAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:45 Zvar(N11V)

VDAKFDKEQQ VAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:46 Zvar(N11A)

VDAKFDKEQQ AAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:47 Zvar(N11H)

VDAKFDKEQQ HAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:48 Zvar(N11R)

VDAKFDKEQQ RAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

SEQ ID NO:49 Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:50 Zvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPK

SEQ ID NO:54 Zvar(Q9A,N11E, A29G,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:55 Zvar(Q9A,N11E, A29S,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNSF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:56 Zvar(Q9A,N11E, A29Y,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNYF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:57 Zvar(Q9A,N11E, A29Q,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNQF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:58 Zvar(Q9A,N11E, A29T,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNTF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:59 Zvar(Q9A,N11E, A29N,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNNF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:60 Zvar(Q9A,N11E, A29F,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNFF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:61 Zvar(Q9A,N11E, A29L,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNLF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:62 Zvar(Q9A,N11E, A29W,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNWF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:63 Zvar(Q9A,N11E, A29I,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNIF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:64 Zvar(Q9A,N11E, A29M,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNMF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:65 Zvar(Q9A,N11E, A29V,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNVF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:66 Zvar(Q9A,N11E, A29D,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNDF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:67 Zvar(Q9A,N11E, A29E,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNEF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:68 Zvar(Q9A,N11E, A29H,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNHF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:69 Zvar(Q9A,N11E, A29R,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNRF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:70 Zvar(Q9A,N11E, A29K,Q40V,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNKF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:71 Zvar(Q9A,N11E, Q40V,A42K,N43A,L44I,D53F)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNFAQAPK

SEQ ID NO:72 Zvar(Q9A,N11E, Q40V,A42K,N43A,L44I,D53Y)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNYAQAPK

SEQ ID NO:73 Zvar(Q9A,N11E, Q40V,A42K,N43A,L44I,D53W)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNWAQAPK

SEQ ID NO:74 Zvar(Q9A,N11E, Q40V,A42K,N43A,L44I,D53K)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNKAQAPK

SEQ ID NO:75 Zvar(Q9A,N11E, Q40V,A42K,N43A,L44I,D53R)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNRAQAPK

SEQ ID NO:76 Zvar(Q9del,N11E, Q40V,A42K,N43A,L44I)

VDAKFDKE_Q EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:77 Zvar(Q9A,N11E, Q40del,A42K,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPS_ SKAILAEAKK LNDAQAPK

SEQ ID NO:78 Zvar(Q9A,N11E, Q40V,A42del,N43A,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV S_AILAEAKK LNDAQAPK

SEQ ID NO:79 Zvar(Q9A,N11E, Q40V,A42K,N43del,L44I)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SK_ILAEAKK LNDAQAPK

SEQ ID NO:89 Zvar(D2del,A3del,K4del,Q9A,N11E,Q40V,A42K,N43A,L44I)

V__FDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:90 Zvar(V1de1,D2de1,Q9A,N11E,Q40V,A42K,N43A,L44I,K58de1)

_AKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAP_

SEQ ID NO:91Zvar(K4del,F5del,D6del,K7del,E8del,Q9A,N11E,Q40V,A42K,N43A,L44I)

VDA___AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:92 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,A56de1,P57de1,K58de1)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQ__

SEQ ID NO:93 Zvar(V1del,,D2del,A3del,Q9A,N11E,Q40V,A42K,N43A,L44I)

__KFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:94Zvar(V1del,D2del,A3del,K4del,F5del,D6del,K7del,E8del,Q9A,N11E,Q40V,A42K,N43A,L44I)

__AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

SEQ ID NO:95 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,K58_insYEDG)

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKYE DG

In a second aspect the present invention discloses a multimercomprising, or consisting essentially of, a plurality of polypeptideunits as defined by any embodiment disclosed above. The use of multimersmay increase the immunoglobulin binding capacity and multimers may alsohave a higher alkali stability than monomers. The multimer can e.g. be adimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, anoctamer or a nonamer. It can be a homomultimer, where all the units inthe multimer are identical or it can be a heteromultimer, where at leastone unit differs from the others. Advantageously, all the units in themultimer are alkali stable, such as by comprising the mutationsdisclosed above. The polypeptides can be linked to each other directlyby peptide bonds between the C-terminal and N-terminal ends of thepolypeptides. Alternatively, two or more units in the multimer can belinked by linkers comprising oligomeric or polymeric species, such aslinkers comprising peptides with up to 25 or 30 amino acids, such as3-25 or 3-20 amino acids. The linkers_may e.g. comprise or consistessentially of a peptide sequence defined by, or having at least 90%identity or at least 95% identity, with an amino acid sequence selectedfrom the group consisting of APKVDAKFDKE (SEQ ID NO:96), APKVDNKFNKE(SEQ ID NO:97), APKADNKFNKE (SEQ ID NO:98), APKVFDKE (SEQ ID NO:99),APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQ ID NO:101), APKVDA (SEQ ID NO:102), VDAKFDKE (SEQ ID NO:103), APKKFDKE (SEQ ID NO: 104), APK,APKYEDGVDAKFDKE (SEQ ID NO: 105) and YEDG (SEQ ID NO: 106) oralternatively selected from the group consisting of APKADNKFNKE (SEQ IDNO:98), APKVFDKE (SEQ ID NO:99), APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQID NO:101), APKVDA (SEQ ID NO: 102), VDAKFDKE (SEQ ID NO:103), APKKFDKE(SEQ ID NO: 104), APKYEDGVDAKFDKE (SEQ ID NO: 105) and YEDG (SEQ IDNO:106). They can also consist essentially of a peptide sequence definedby or having at least 90% identity or at least 95% identity with anamino acid sequence selected from the group consisting of APKADNKFNKE(SEQ ID NO:98), APKVFDKE (SEQ ID NO:99), APAKFDKE (SEQ ID NO: 100),AKFDKE (SEQ ID NO:101), APKVDA (SEQ ID NO: 102), VDAKFDKE (SEQ IDNO:103), APKKFDKE (SEQ ID NO: 104), APK and APKYEDGVDAKFDKE (SEQ ID NO:105). In some embodiments the linkers do not consist of the peptidesAPKVDAKFDKE (SEQ ID NO:96) or APKVDNKFNKE (SEQ ID NO:97), oralternatively do not consist of the peptides APKVDAKFDKE (SEQ ID NO:96),APKVDNKFNKE (SEQ ID NO:97).

The nature of such a linker should preferably not destabilize thespatial conformation of the protein units. This can e.g. be achieved byavoiding the presence of proline in the linkers. Furthermore, saidlinker should preferably also be sufficiently stable in alkalineenvironments not to impair the properties of the mutated protein units.For this purpose, it is advantageous if the linkers do not containasparagine. It can additionally be advantageous if the linkers do notcontain glutamine. The multimer may further at the N-terminal endcomprise a plurality of amino acid residues e.g. originating from thecloning process or constituting a residue from a cleaved off signalingsequence. The number of additional amino acid residues may e.g. be 20 orless, such as 15 or less, such as 10 or less or 5 or less. As a specificexample, the multimer may comprise an AQ, AQGT (SEQ ID NO:107), VDAKFDKE(SEQ ID NO:103), AQVDAKFDKE (SEQ ID NO:108) or AQGTVDAKFDKE (SEQ IDNO:109) sequence at the N-terminal end.

In certain embodiments, the multimer may comprise, or consistessentially, of a sequence selected from the group consisting of: SEQ IDNO:80-87. These and additional sequences are listed below and named asParent(Mutations)n, where n is the number of monomer units in amultimer.

SEQ ID NO:17 Zvar(Q9A,N11E,N43A)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQSAALLAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAFIQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKC

SEQ ID NO:18 Zvar(Q9A,N11E,N28A,N43A)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQSAALLAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAFIQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPKC

SEQ ID NO:19 Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKKLNDAQAPKC

SEQ ID NO:20 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPKC

SEQ ID NO:30 Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)4

AQGT VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKRYNDAQAPK VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQSRAILAEAKR YNDAQAPK VDAKFDKEQQ KAFYEILKLP NLTEEQRNAFIQKLKDEPSQ SRAILAEAKR YNDAQAPK VDAKFDKEQQ KAFYEILKLPNLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKC

SEQ ID NO:31 Zvar(Q9A,N11K,H18K,D37E,A42R)4

AQGT VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKKLNDAQAPK VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQSRNLLAEAKK LNDAQAPK VDAKFDKEAQ KAFYEILKLP NLTEEQRNAFIQSLKDEPSQ SRNLLAEAKK LNDAQAPK VDAKFDKEAQ KAFYEILKLPNLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKC

SEQ ID NO:32 Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSVSKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAFIQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:33 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)6

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPKC

SEQ ID NO:34 Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSVSKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAFIQSLKDEPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:35 Zvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)4

AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSVSRAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAFIQSLKDEPSV SRAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKC

SEQ ID NO:80 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with D2, A3 and K4 inlinker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:81 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with K58, V1 and D2 inlinker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAP AKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:82 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with P57, K58, V1, D2and A3 in linker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAP AKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:83 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with K4, F5, D6, K7 andE8 in linker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:84 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with A56, P57 and K58in linker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:85 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with V1, D2 and A3 inlinker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK KFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:86 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with V1, D2, A3, K4,F5, D6, K7 and E8 in linker deleted

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

SEQ ID NO:87 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)2 with YEDG inserted inlinker between K58 and V1

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK YEDG VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSVSKAILAEAKK LNDAQAPKC

SEQ ID NO:88 Zvar2

VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSVSKAILAEAKK LNDAQAPKC

In some embodiments, the polypeptide and/or multimer, as disclosedabove, further comprises at the C-terminal or N-terminal end one or morecoupling elements, selected from the group consisting of one or morecysteine residues, a plurality of lysine residues and a plurality ofhistidine residues. The coupling element(s) may also be located within1-5 amino acid residues, such as within 1-3 or 1-2 amino acid residuesfrom the C-terminal or N-terminal end. The coupling element may e.g. bea single cysteine at the C-terminal end. The coupling element(s) may bedirectly linked to the C- or N-terminal end, or it/they may be linkedvia a stretch comprising up to 15 amino acids, such as 1-5, 1-10 or 5-10amino acids. This stretch should preferably also be sufficiently stablein alkaline environments not to impair the properties of the mutatedprotein. For this purpose, it is advantageous if the stretch does notcontain asparagine. It can additionally be advantageous if the stretchdoes not contain glutamine. An advantage of having a C-terminal cysteineis that endpoint coupling of the protein can be achieved throughreaction of the cysteine thiol with an electrophilic group on a support.This provides excellent mobility of the coupled protein which isimportant for the binding capacity.

The alkali stability of the polypeptide or multimer can be assessed bycoupling it to an SPR chip, e.g. to Biacore CM5 sensor chips asdescribed in the examples, using e.g. NHS- or maleimide couplingchemistries, and measuring the immunoglobulin-binding capacity of thechip, typically using polyclonal human IgG, before and after incubationin alkaline solutions at a specified temperature, e.g. 22 +/- 2° C. Theincubation can e.g. be performed in 0.5 M NaOH for a number of 10 mincycles, such as 100, 200 or 300 cycles. The IgG capacity of the matrixafter 100 10 min incubation cycles in 0.5 M NaOH at 22 +/- 2° C. can beat least 55, such as at least 60, at least 80 or at least 90% of the IgGcapacity before the incubation. Alternatively, the remaining IgGcapacity after 100 cycles for a particular mutant measured as above canbe compared with the remaining IgG capacity for the parentalpolypeptide/multimer. In this case, the remaining IgG capacity for themutant may be at least 105%, such as at least 110%, at least 125%, atleast 150% or at least 200% of the parental polypeptide/multimer.

In a third aspect the present invention discloses a nucleic acidencoding a polypeptide or multimer according to any embodiment disclosedabove. Thus, the invention encompasses all forms of the present nucleicacid sequence such as the RNA and the DNA encoding the polypeptide ormultimer. The invention embraces a vector, such as a plasmid, which inaddition to the coding sequence comprises the required signal sequencesfor expression of the polypeptide or multimer according the invention.In one embodiment, the vector comprises nucleic acid encoding a multimeraccording to the invention, wherein the separate nucleic acids encodingeach unit may have homologous or heterologous DNA sequences.

In a fourth aspect the present invention discloses an expression system,which comprises, a nucleic acid or a vector as disclosed above. Theexpression system may e.g. be a gram-positive or gram-negativeprokaryotic host cell system, e.g. E.coli or Bacillus sp. which has beenmodified to express the present polypeptide or multimer. In analternative embodiment, the expression system is a eukaryotic host cellsystem, such as a yeast, e.g. Pichia pastoris or Saccharomycescerevisiae, or mammalian cells, e.g. CHO cells.

In a fifth aspect, the present invention discloses a separation matrix,wherein a plurality of polypeptides or multimers according to anyembodiment disclosed above have been coupled to a solid support. Theseparation matrix may comprise at least 11, such as 11-21, 15-21 or15-18 mg/ml Fc-binding ligands covalently coupled to a porous support,wherein:

-   a) the ligands comprise multimers of alkali-stabilized Protein A    domains,-   b) the porous support comprises cross-linked polymer particles    having a volume-weighted median diameter (d50,v) of 56-70, such as    56-66, micrometers and a dry solids weight of 55-80, such as 60-78    or 65-78, mg/ml. The cross-linked polymer particles may further have    a pore size corresponding to an inverse gel filtration    chromatography Kd value of 0.69-0.85, such as 0.70-0.85 or    0.69-0.80, for dextran of Mw 110 kDa. Suitably, the cross-linked    polymer particles can have a high rigidity, to be able to withstand    high flow rates. The rigidity can be measured with a pressure-flow    test as further described in Example 8, where a column packed with    the matrix is subjected to increasing flow rates of distilled water.    The pressure is increased stepwise and the flow rate and back    pressure measured, until the flow rate starts to decrease with    increasing pressures. The maximum flow rate achieved and the maximum    pressure (the back pressure corresponding to the maximum flow rate)    are measured and used as measures of the rigidity. When measured in    a FINELINE 35 column (GE Healthcare Life Sciences) at a bed height    of 300 +/- 10 mm, the max pressure can suitably be at least 0.58    MPa, such as at least 0.60 MPa. This allows for the use of smaller    particle diameters, which is beneficial for the dynamic capacity.    The multimers may e.g. comprise tetramers, pentamers, hexamers or    heptamers of alkali-stabilized Protein A domains, such as hexamers    of alkali-stabilized Protein A domains. The combination of the high    ligand contents with the particle size range, the dry solids weight    range and the optional Kd range provides for a high binding    capacity, e.g. such that the 10% breakthrough dynamic binding    capacity for IgG is at least 45 mg/ml, such as at least 50 or at    least 55 mg/ml at 2.4 min residence time. Alternatively, or    additionally, the 10% breakthrough dynamic binding capacity for IgG    may be at least 60 mg/ml, such as at least 65, at least 70 or at    least 75 mg/ml at 6 min residence time.

The alkali-stabilized Protein A multimers are highly selective for IgGand the separation matrix can suitably have a dissociation constant forhuman IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in 20 mMphosphate buffer, 180 mM NaCl, pH 7.5. This can be determined accordingto the adsorption isotherm method described in N Pakiman et al: J ApplSci 12, 1136-1141 (2012).

In certain embodiments the alkali-stabilized Protein A domains comprisemutants of a parental Fc-binding domain of Staphylococcus Protein A(SpA), as defined by, or having at least 80% such as at least 90%, 95%or 98% identity to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO:51 orSEQ ID NO:52, wherein at least the asparagine or serine residue at theposition corresponding to position 11 in SEQ ID NO:4-7 has been mutatedto an amino acid selected from the group consisting of glutamic acid,lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine,tryptophan, methionine, valine, alanine, histidine and arginine, such asan amino acid selected from the group consisting of glutamic acid andlysine. The amino acid residue at the position corresponding to position40 in SEQ ID NO:4-7 may further be, or be mutated to, a valine. Thealkali-stabilized Protein A domains may also comprise any mutations asdescribed in the polypeptide and/or multimer embodiments above.

In some embodiments the alkali-stabilized Protein A domains comprise anFc-binding polypeptide having an amino acid sequence as defined by, orhaving at least 80% or at least 90, 95% or 98% identity to SEQ ID NO:53.

X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉ SX₁₀X₁₁X₁₂LAEAKX₁₃X₁₄NX₁₅AQ (SEQ ID NO:53)

wherein individually of each other:

-   X₁=A or Q or is deleted-   X₂=E,K,Y,T,F,L,W,I,M,V,A,H or R-   X₃=H or K-   X₄=A or N-   X₅=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K-   X₆=Q or E-   X₇=S or K-   X₈=E or D-   X₉=Q or V or is deleted-   X₁₀=K,R or A or is deleted-   X₁₁=A,E or N or is deleted-   X₁₂=I or L-   X₁₃=K or R-   X₁₄=L or Y-   X₁₅=D, F,Y,W,K or R

In some embodiments, the amino acid residues may individually of eachother be:

-   a) X₁ = A or is deleted, X₂ = E, X₃ = H, X₄ = N, X₆ = Q, X₇ = S, X₈    = D, X₉ = V or is deleted, X₁₀ = K or is deleted, X₁₁ = A or is    deleted, X₁₂ = I, X₁₃ = K, X₁₄ = L.-   b) X₁=A, X₂ = E, X₃ = H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ =    V, X₁₀ = K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = Land X₁₅=D.-   c) X₁ is A, X₂ = E, X₃ = H, X₄ = N, X₆ = Q, X₇ = S, X₈ = D, X₉ = V,    X₁₀ = K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = Land X₁₅=D or-   d) X₁ is A, X₃ = H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V,    X₁₀ = K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = Land X₁₅=D.

In certain embodiments the invention discloses a separation matrixcomprising at least 15, such as 15-21 or 15-18 mg/ml Fc-binding ligandscovalently coupled to a porous support, wherein the ligands comprisemultimers of alkali-stabilized Protein A domains. These multimers cansuitably be as disclosed in any of the embodiments described above or asspecified below.

Such a matrix is useful for separation of immunoglobulins or otherFc-containing proteins and, due to the improved alkali stability of thepolypeptides/multimers, the matrix will withstand highly alkalineconditions during cleaning, which is essential for long-term repeateduse in a bioprocess separation setting. The alkali stability of thematrix can be assessed by measuring the immunoglobulin-binding capacity,typically using polyclonal human IgG, before and after incubation inalkaline solutions at a specified temperature, e.g. 22 +/- 2° C. Theincubation can e.g. be performed in 0.5 M or 1.0 M NaOH for a number of15 min cycles, such as 100, 200 or 300 cycles, corresponding to a totalincubation time of 25, 50 or 75 h. The IgG capacity of the matrix after96-100 15 min incubation cycles or a total incubation time of 24 or 25 hin 0.5 M NaOH at 22 +/- 2° C. can be at least 80, such as at least 85,at least 90 or at least 95% of the IgG capacity before the incubation.The capacity of the matrix after a total incubation time of 24 h in 1.0M NaOH at 22 +/- 2° C. can be at least 70, such as at least 80 or atleast 90% of the IgG capacity before the incubation. The the 10%breakthrough dynamic binding capacity (Qb10%) for IgG at 2.4 min or 6min residence time may e.g. be reduced by less than 20 % afterincubation 31 h in 1.0 M aqueous NaOH at 22 +/- 2 C.

As the skilled person will understand, the expressed polypeptide ormultimer should be purified to an appropriate extent before beingimmobilized to a support. Such purification methods are well known inthe field, and the immobilization of protein-based ligands to supportsis easily carried out using standard methods. Suitable methods andsupports will be discussed below in more detail.

The solid support of the matrix according to the invention can be of anysuitable well-known kind. A conventional affinity separation matrix isoften of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carboxamido (—CONH₂, possibly in N— substituted forms),amiNO:(-NH₂, possibly in substituted form), oligo- or polyethylenoxygroups on their external and, if present, also on internal surfaces. Thesolid support can suitably be porous. The porosity can be expressed as aKav or Kd value (the fraction of the pore volume available to a probemolecule of a particular size) measured by inverse size exclusionchromatography, e.g. according to the methods described in GelFiltration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp6-13. Kav is determined as the ratio (V_(e)-V₀)/(V_(t)-V₀), where Ve isthe elution volume of a probe molecule (e.g. Dextran 110 kD), V₀ is thevoid volume of the column (e.g. the elution volume of a high Mw voidmarker, such as raw dextran) and Vt is the total volume of the column.Kd can be determined as (V_(e)-V₀)/V_(i), where V_(i) is the elutionvolume of a salt (e.g. NaCl) able to access all the volume except thematrix volume (the volume occupied by the matrix polymer molecules). Bydefinition, both Kd and Kav values always lie within the range 0 - 1.The Kav value can advantageously be 0.6 - 0.95, e.g. 0.7 - 0.90 or 0.6 -0.8, as measured with dextran of Mw 110 kDa as a probe molecule. The Kdvalue as measured with dextran of Mw 110 kDa can suitably be 0.68-0.90,such as 0.68-0.85 or 0.70-0.85. An advantage of this is that the supporthas a large fraction of pores able to accommodate both thepolypeptides/multimers of the invention and immunoglobulins binding tothe polypeptides/multimers and to provide mass transport of theimmunoglobulins to and from the binding sites.

The polypeptides or multimers may be attached to the support viaconventional coupling techniques utilising e.g. thiol, amiNO:and/orcarboxy groups present in the ligand. Bisepoxides, epichlorohydrin,CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents.Between the support and the polypeptide/multimer, a molecule known as aspacer can be introduced, which improves the availability of thepolypeptide/multimer and facilitates the chemical coupling of thepolypeptide/multimer to the support. Depending on the nature of thepolypeptide/multimer and the coupling conditions, the coupling may be amultipoint coupling (e.g. via a plurality of lysines) or a single pointcoupling (e.g. via a single cysteine). Alternatively, thepolypeptide/multimer may be attached to the support by non-covalentbonding, such as physical adsorption or biospecific adsorption.

In some embodiments the matrix comprises 5 - 25, such as 5-20 mg/ml, 5 -15 mg/ml, 5 - 11 mg/ml or 6 - 11 mg/ml of the polypeptide or multimercoupled to the support. The amount of coupled polypeptide/multimer canbe controlled by the concentration of polypeptide/multimer used in thecoupling process, by the activation and coupling conditions used and/orby the pore structure of the support used. As a general rule theabsolute binding capacity of the matrix increases with the amount ofcoupled polypeptide/multimer, at least up to a point where the poresbecome significantly constricted by the coupled polypeptide/multimer.Without being bound by theory, it appears though that for the Kd valuesrecited for the support, the constriction of the pores by coupled ligandis of lower significance. The relative binding capacity per mg coupledpolypeptide/multimer will decrease at high coupling levels, resulting ina cost-benefit optimum within the ranges specified above.

In certain embodiments the polypeptides or multimers are coupled to thesupport via thioether bonds. Methods for performing such coupling arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment. Thioether bonds areflexible and stable and generally suited for use in affinitychromatography. In particular when the thioether bond is via a terminalor near-terminal cysteine residue on the polypeptide or multimer, themobility of the coupled polypeptide/multimer is enhanced which providesimproved binding capacity and binding kinetics. In some embodiments thepolypeptide/multimer is coupled via a C-terminal cysteine provided onthe protein as described above. This allows for efficient coupling ofthe cysteine thiol to electrophilic groups, e.g. epoxide groups,halohydrin groups etc. on a support, resulting in a thioether bridgecoupling.

In certain embodiments the support comprises a polyhydroxy polymer, suchas a polysaccharide. Examples of polysaccharides include e.g. dextran,starch, cellulose, pullulan, agar, agarose etc. Polysaccharides areinherently hydrophilic with low degrees of nonspecific interactions,they provide a high content of reactive (activatable) hydroxyl groupsand they are generally stable towards alkaline cleaning solutions usedin bioprocessing.

In some embodiments the support comprises agar or agarose. The supportsused in the present invention can easily be prepared according tostandard methods, such as inverse suspension gelation (S Hjertén:Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the basematrices are commercially available products, such as crosslinkedagarose beads sold under the name of SEPHAROSE™ FF (GE Healthcare). Inan embodiment, which is especially advantageous for large-scaleseparations, the support has been adapted to increase its rigidity usingthe methods described in US6602990 or US7396467, which are herebyincorporated by reference in their entireties, and hence renders thematrix more suitable for high flow rates.

In certain embodiments the support, such as a polymer, polysaccharide oragarose support, is crosslinked, such as with hydroxyalkyl ethercrosslinks. Crosslinker reagents producing such crosslinks can be e.g.epihalohydrins like epichlorohydrin, diepoxides like butanedioldiglycidyl ether, allylating reagents like allyl halides or allylglycidyl ether. Crosslinking is beneficial for the rigidity of thesupport and improves the chemical stability. Hydroxyalkyl ethercrosslinks are alkali stable and do not cause significant nonspecificadsorption.

Alternatively, the solid support is based on synthetic polymers, such aspolyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers are easily produced accordingto standard methods, see e.g. “Styrene based polymer supports developedby suspension polymerization” (R Arshady: Chimica e L′Industria 70(9),70-75 (1988)).

Alternatively, a commercially available product, such as SOURCE™ (GEHealthcare) is used. In another alternative, the solid support accordingto the invention comprises a support of inorganic nature, e.g. silica,zirconium oxide etc.

In yet another embodiment, the solid support is in another form such asa surface, a chip, capillaries, or a filter (e.g. a membrane or a depthfilter matrix).

As regards the shape of the matrix according to the invention, in oneembodiment the matrix is in the form of a porous monolith. In analternative embodiment, the matrix is in beaded or particle form thatcan be porous or non-porous. Matrices in beaded or particle form can beused as a packed bed or in a suspended form. Suspended forms includethose known as expanded beds and pure suspensions, in which theparticles or beads are free to move. In case of monoliths, packed bedand expanded beds, the separation procedure commonly followsconventional chromatography with a concentration gradient. In case ofpure suspension, batch-wise mode will be used.

In a sixth aspect, the present invention discloses a method of isolatingan immunoglobulin, wherein a separation matrix as disclosed above isused. The method may comprise the steps of:

-   a) contacting a liquid sample comprising an immunoglobulin with a    separation matrix as disclosed above,-   b) washing the separation matrix with a washing liquid,-   c) eluting the immunoglobulin from the separation matrix with an    elution liquid, and-   d) cleaning the separation matrix with a cleaning liquid, which may    comprise 0.1 - 1.0 M NaOH or KOH, such as 0.4 - 1.0 M NaOH or KOH.

Steps a) - d) may be repeated at least 10 times, such as at least 50times or 50 - 200 times.

In certain embodiments, the method comprises the steps of:

-   a) contacting a liquid sample comprising an immunoglobulin with a    separation matrix as disclosed above,-   b) washing said separation matrix with a washing liquid,-   c) eluting the immunoglobulin from the separation matrix with an    elution liquid, and-   d) cleaning the separation matrix with a cleaning liquid, which can    alternatively be called a cleaning-in-place (CIP) liquid, e.g. with    a contact (incubation) time of at least 10 min.

The method may also comprise steps of, before step a), providing anaffinity separation matrix according to any of the embodiments describedabove and providing a solution comprising an immunoglobulin and at leastone other substance as a liquid sample and of, after step c), recoveringthe eluate and optionally subjecting the eluate to further separationsteps, e.g. by anion or cation exchange chromatography, multimodalchromatography and/or hydrophobic interaction chromatography. Suitablecompositions of the liquid sample, the washing liquid and the elutionliquid, as well as the general conditions for performing the separationare well known in the art of affinity chromatography and in particularin the art of Protein A chromatography. The liquid sample comprising anFc-containing protein and at least one other substance may comprise hostcell proteins (HCP), such as CHO cell, E.coli or yeast proteins.Contents of CHO cell and E.coli proteins can conveniently be determinedby immunoassays directed towards these proteins, e.g. the CHO HCP orE.coli HCP ELISA kits from Cygnus Technologies. The host cell proteinsor CHO cell/E.coli proteins may be desorbed during step b).

The elution may be performed by using any suitable solution used forelution from Protein A media. This can e.g. be a solution or buffer withpH 5 or lower, such as pH 2.5 - 5 or 3 - 5. It can also in some cases bea solution or buffer with pH 11 or higher, such as pH 11 - 14 or pH 11 -13. In some embodiments the elution buffer or the elution buffergradient comprises at least one mono- di- or trifunctional carboxylicacid or salt of such a carboxylic acid. In certain embodiments theelution buffer or the elution buffer gradient comprises at least oneanion species selected from the group consisting of acetate, citrate,glycine, succinate, phosphate, and formiate.

In some embodiments, the cleaning liquid is alkaline, such as with a pHof 13 - 14. Such solutions provide efficient cleaning of the matrix, inparticular at the upper end of the interval

In certain embodiments, the cleaning liquid comprises 0.1 - 2.0 M NaOHor KOH, such as 0.5 - 2.0 or 0.5 - 1.0 M NaOH or KOH. These areefficient cleaning solutions, and in particular so when the NaOH or KOHconcentration is above 0.1 M or at least 0.5 M. The high stability ofthe polypeptides of the invention enables the use of such stronglyalkaline solutions.

The method may also include a step of sanitizing the matrix with asanitization liquid, which may e.g. comprise a peroxide, such ashydrogen peroxide and/or a peracid, such as peracetic acid or performicacid.

In some embodiments, steps a) - d) are repeated at least 10 times, suchas at least 50 times, 50 - 200, 50-300 or 50-500 times. This isimportant for the process economy in that the matrix can be re-used manytimes.

Steps a) - c) can also be repeated at least 10 times, such as at least50 times, 50 - 200, 50-300 or 50-500 times, with step d) being performedafter a plurality of instances of step c), such that step d) isperformed at least 10 times, such as at least 50 times. Step d) can e.g.be performed every second to twentieth instance of step c).

EXAMPLES Mutagenesis of Protein

Site-directed mutagenesis was performed by a two-step PCR usingoligonucleotides coding for the mutations. As template a plasmidcontaining a single domain of either Z, B or C was used. The PCRfragments were ligated into an E. coli expression vector. DNA sequencingwas used to verify the correct sequence of inserted fragments.

To form multimers of mutants an Acc I site located in the startingcodons (GTA GAC) of the B, C or Z domain was used, corresponding toamino acids VD. The vector for the monomeric domain was digested withAcc I and phosphatase treated. Acc I sticky-ends primers were designed,specific for each variant, and two overlapping PCR products weregenerated from each template. The PCR products were purified and theconcentration was estimated by comparing the PCR products on a 2%agarose gel. Equal amounts of the pair wise PCR products were hybridized(90° C. -> 25° C. in 45 min) in ligation buffer. The resulting productconsists approximately to ¼ of fragments likely to be ligated into anAcc I site (correct PCR fragments and/or the digested vector). Afterligation and transformation colonies were PCR screened to identifyconstructs containing the desired mutant. Positive clones were verifiedby DNA sequencing.

Construct Expression and Purification

The constructs were expressed in the bacterial periplasm by fermentationof E. coli K12 in standard media. After fermentation the cells wereheat-treated to release the periplasm content into the media. Theconstructs released into the medium were recovered by microfiltrationwith a membrane having a 0.2 µm pore size.

Each construct, now in the permeate from the filtration step, waspurified by affinity. The permeate was loaded onto a chromatographymedium containing immobilized IgG (IgG Sepharose 6FF, GE Healthcare).The loaded product was washed with phosphate buffered saline and elutedby lowering the pH.

The elution pool was adjusted to a neutral pH (pH 8) and reduced byaddition of dithiothreitol. The sample was then loaded onto an anionexchanger. After a wash step the construct was eluted in a NaCl gradientto separate it from any contaminants. The elution pool was concentratedby ultrafiltration to 40-50 mg/ml. It should be noted that thesuccessful affinity purification of a construct on an immobilized IgGmedium indicates that the construct in question has a high affinity toIgG.

The purified ligands were analyzed with RPC LC-MS to determine thepurity and to ascertain that the molecular weight corresponded to theexpected (based on the amino acid sequence).

Example 1

The purified monomeric ligands listed in Table 1, further comprising forSEQ ID NO:8-16, 23-28 and 36-48 an AQGT leader sequence at theN-terminus and a cysteine at the C terminus, were immobilized on BiacoreCM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kitof GE Healthcare (for carbodiimide coupling of amines on thecarboxymethyl groups on the chip) in an amount sufficient to give asignal strength of about 200-1500 RU in a Biacore surface plasmonresonance (SPR) instrument (GE Healthcare, Sweden) . To follow the IgGbinding capacity of the immobilized surface 1 mg/ml human polyclonal IgG(Gammanorm) was flowed over the chip and the signal strength(proportional to the amount of binding) was noted. The surface was thencleaned-in-place (CIP), i.e. flushed with 500 mM NaOH for 10 minutes atroom temperature (22 +/- 2° C.). This was repeated for 96-100 cycles andthe immobilized ligand alkaline stability was followed as the remainingIgG binding capacity (signal strength) after each cycle. The results areshown in Table 1 and indicate that at least the ligands Zvar(N11K)1,Zvar(N11E)1, Zvar(N11Y)1, Zvar(N11T)1, Zvar(N11F)1, Zvar(N11L)1,Zvar(N11W)1, ZN11I)1, Zvar(N11M)1, Zvar(N11V)1, Zvar(N11A)1,Zvar(N11H1), Zvar(N11R)1, Zvar(N11E,Q32A)1, Zvar(N11E,Q32E,Q40E)1 andZvar(N11E,Q32E,K50R)1, Zvar(Q9A,N11E,N43A)1, Zvar(Q9A,N11E,N28A,N43A)1,Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)1,Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1,Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)1,Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,E51Y)1,Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1, Zvar(N11K,H18K, D37E, A42R, N43A, L44I)1, Zvar(Q9A, N11K, H18K, D37E, A42R, N43A,L44I)1 and Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)1, as wellas the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 havingG,S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K in position 29, the varieties ofZvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 having F,Y,W,K or R in position 53and the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 where Q9, Q40,A42 or N43 has been deleted, have an improved alkali stability comparedto the parental structure Zvar1, used as the reference. Further, theligands B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 and C(Q9A,N11E,E43A)1 have animproved stability compared to the parental B and C domains, used asreferences.

TABLE 1 Monomeric ligands, evaluated by Biacore (0.5 M NaOH) LigandSequence Capacity after 96-100 cycles Reference capacity after 96-100cycles Capacity relative to reference Zvar(N11E,Q32A)1 SEQ ID NO:12 57%55% 1.036 Zvar(N11E)1 SEQ ID NO:13 59% 55% 1.073 Zvar(N11E,Q32E,Q40E)1SEQ ID NO:14 52% 51% 1.020 Zvar(N11E,Q32E,K50R)1 SEQ ID NO:15 53% 51%1.039 Zvar(N11K)1 SEQ ID NO:16 62% 49% 1.270 Zvar(N11Y)1 SEQ ID NO:3855% 46% 1.20 Zvar(N11T)1 SEQ ID NO:39 50% 46% 1.09 Zvar(N11F)1 SEQ IDNO:40 55% 46% 1.20 Zvar(N11L)1 SEQ ID NO:41 57% 47% 1.21 Zvar(N11W)1 SEQID NO:42 57% 47% 1.21 Zvar(N11I)1 SEQ ID NO:43 57% 47% 1.21 Zvar(N11M)1SEQ ID NO:44 58% 46% 1.26 Zvar(N11V)1 SEQ ID NO:45 56% 46% 1.22Zvar(N11A)1 SEQ ID NO:46 58% 46% 1.26 Zvar(N11H)1 SEQ ID NO:47 57% 46%1.24 Zvar(N11R)1 SEQ ID NO:48 59% 46% 1.28 Zvar(Q9A,N11E,N43A)1 SEQ IDNO:8 70% 47% 1.49 Zvar(Q9A,N11E,N28A,N43A)1 SEQ ID NO:9 68% 47% 1.45Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)1 SEQ ID NO:10 67% 47% 1.43Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID NO: 11 66% 47% 1.40Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)1 SEQ ID NO:24 65% 48% 1.35Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1 SEQ ID NO:23 67% 46%1.46 Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,KSOR,L51Y)1 SEQ IDNO:25 59% 46% 1.28 Zvar(N11K, H18K, D37E, A42R, N43A, L44I)1 SEQ IDNO:26 59% 45% 1.31 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)1 SEQ IDNO:27 63% 45% 1.40 Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)1SEQ ID NO:28 67% 45% 1.49 B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID NO:3639% 35% 1.11 C(Q9A,N11E,E43A)1 SEQ ID NO:37 60% 49% 1.22Zvar(Q9A,N11E,A29G,Q40V,A42K,N43A,L44I)1 SEQ ID NO:54 69% 48% 1.44Zvar(Q9A,N11E,A29S,Q40V,A42K,N43A,L44I)1 SEQ ID NO:55 66% 48% 1.38Zvar(Q9A,N11E,A29Y,Q40V,A42K,N43A,L44I)1 SEQ ID NO:56 61% 48% 1.27Zvar(Q9A,N11E,A29Q,Q40V,A42K,N43A,L44I)1 SEQ ID NO:57 60% 47% 1.28Zvar(Q9A,N11E,A29T,Q40V,A42K,N43A,L44I)1 SEQ ID NO:58 60% 47% 1.28Zvar(Q9A,N11E,A29N,Q40V,A42K,N43A,L44I)1 SEQ ID NO:59 61% 47% 1.30Zvar(Q9A,N11E,A29F,Q40V,A42K,N43A,L44I)1 SEQ ID NO:60 62% 46% 1.35Zvar(Q9A,N11E,A29L,Q40V,A42K,N43A,L44I)1 SEQ ID NO:61 61% 46% 1.33Zvar(Q9A,N11E,A29W,Q40V,A42K,N43A,L44I)1 SEQ ID NO:62 60% 46% 1.30Zvar(Q9A,N11E,A29I,Q40V,A42K,N43A,L44I)1 SEQ ID NO:63 58% 47% 1.23Zvar(Q9A,N11E,A29M,Q40V,A42K,N43A,L44I)1 SEQ ID NO:64 62% 47% 1.32Zvar(Q9A,N11E,A29V,Q40V,A42K,N43A,L44I)1 SEQ ID NO:65 62% 47% 1.32Zvar(Q9A,N11E,A29D,Q40V,A42K,N43A,L44I)1 SEQ ID NO:66 56% 47% 1.19Zvar(Q9A,N11E,A29E,Q40V,A42K,N43A,L44I)1 SEQ ID NO:67 57% 47% 1.21Zvar(Q9A,N11E,A29H,Q40V,A42K,N43A,L44I)1 SEQ ID NO:68 57% 47% 1.21Zvar(Q9A,N11E,A29R,Q40V,A42K,N43A,L44I)1 SEQ ID NO:69 58% 46% 1.26Zvar(Q9A,N11E,A29K,Q40V,A42K,N43A,L44I)1 SEQ ID NO:70 59% 46% 1.28Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53F)1 SEQ ID NO:71 58% 46% 1.26Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53Y)1 SEQ ID NO:72 59% 46% 1.28Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53W)1 SEQ ID NO:73 62% 46% 1.35Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53K)1 SEQ ID NO:74 65% 46% 1.41Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53R)1 SEQ ID NO:75 60% 46% 1.30Zvar(Q9del,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID NO:76 60% 46% 1.30Zvar(Q9A,N11 E,Q40del,A42K,N43A,L44I)1 SEQ ID NO:77 59% 46% 1.28Zvar(Q9A,N11E,Q40V,A42del,N43A,L44I)1 SEQ ID NO:78 57% 46% 1.24Zvar(Q9A,N11E,Q40V,A42K,N43del,L44I)1 SEQ ID NO:79 55% 46% 1.20

The Biacore experiment can also be used to determine the binding anddissociation rates between the ligand and IgG. This was used with theset-up as described above and with an IgG1 monoclonal antibody as probemolecule. For the reference Zvar1, the on-rate (10⁵ M⁻¹s⁻¹) was 3.1 andthe off-rate (10⁵ s⁻¹) was 22.1, giving an affinity (off-rate/on-rate)of 713 pM. For Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 (SEQ ID NO:11), theon-rate was 4.1 and the off-rate 43.7, with affinity 1070 pM. The IgGaffinity was thus somewhat higher for the mutated variant.

Example 2

The purified dimeric, tetrameric and hexameric ligands listed in Table 2were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden),using the amine coupling kit of GE Healthcare (for carbodiimide couplingof amines on the carboxymethyl groups on the chip) in an amountsufficient to give a signal strength of about 200-1500 RU in a Biacoreinstrument (GE Healthcare, Sweden) . To follow the IgG binding capacityof the immobilized surface 1 mg/ml human polyclonal IgG (Gammanorm) wasflowed over the chip and the signal strength (proportional to the amountof binding) was noted. The surface was then cleaned-in-place (CIP), i.e.flushed with 500 mM NaOH for 10 minutes at room temperature (22 +/- 2°C.). This was repeated for 300 cycles and the immobilized ligandalkaline stability was followed as the remaining IgG binding capacity(signal strength) after each cycle. The results are shown in Table 2 andin FIG. 2 and indicate that at least the ligands Zvar(Q9A,N11E,N43A)4,Zvar(Q9A,N11E,N28A,N43A)4, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4 andZvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4,Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)4 andZvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)4 have an improved alkalistability compared to the parental structure Zvar4, which was used as areference. The hexameric ligand Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)6 alsohas improved alkali stability compared to the parental structure Zvar6,used as a reference. Further, Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I) dimerswith deletions of a) D2,A3,K4; b) K58,V1,D2; c) P57,K58,V1,D2,A3; d)K4,F5,D6,K7,E8; e) A56,P57,K58; V1,D2,A3 or f) V1,D2,A3,K4,F5,D6,K7,E8from the linker region between the two monomer units have improvedalkali stability compared to the parental structure Zvar2, used as areference. Also Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I) dimers with aninsertion of YEDG between K58 and V1 in the linker region have improvedalkali stability compared to Zvar2.

TABLE 2 Dimeric, tetrameric and hexameric ligands, evaluated by Biacore(0.5 M NaOH) Ligand SEQ ID NO: Remaining capacity 100 cycles (%)Capacity relative to ref. 100 cycles Remaining capacity 200 cycles (%)Capacity relative to ref. 200 cycles Remaining capacity 300 cycles (%)Capacity relative to ref. 300 cycles Zvar4 21 67 1 36 1 16 1Zvar(Q9A,N11E,N43A)4 17 81 1.21 62 1.72 41 2.56 Zvar(Q9A,N11E,N28A,N43A)4 18 80 1.19 62 1.72 42 2.62 Zvar(Q9A,N11E,Q40V,A 42K,N43E,L44I)4 1984 1.25 65 1.81 48 3.00 Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)4 20 90 1.3474 2.06 57 3.56 Zvar(Q9A,N11E,N28A,Q 40V,A42K,N43A,L44I)4 32 84 1.24 Nottested Not tested Not tested Not tested Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)6 33 87 1.30 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,D37E,Q 40V,A42K,N43A,L44I)4 34 81 1.13 Not tested Nottested Not tested Not tested Zvar(Q9A,N11E,D37E,Q 40V,A42R,N43A,L44I)435 84 1.17 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with D2, A3 and K4 in linkerdeleted 80 70 1.27 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with K58, V1 and D2 in linkerdeleted 81 76 1.38 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with P57, K58, V1, D2 and A3 inlinker deleted 82 74 1.35 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with K4, F5, D6, K7 and E8 inlinker deleted 83 70 1.30 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with A56, P57 and K58 in linkerdeleted 84 68 1.26 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with V1, D2 and A3 in linkerdeleted 85 75 1.39 Not tested Not tested Not tested Not testedZvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with V1, D2, A3, K4, F5, D6, K7 andE8 in linker deleted 86 62 1.13 Not tested Not tested Not tested Nottested Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with YEDG inserted in linkerbetween K58 and V1 87 72 1.31 Not tested Not tested Not tested Nottested Zvar2 88 55 1 Not tested Not tested Not tested Not tested

Example 3

Example 2 was repeated with 100 CIP cycles of three ligands using 1 MNaOH instead of 500 mM as in Example 2. The results are shown in Table 3and show that all three ligands have an improved alkali stability alsoin 1 M NaOH, compared to the parental structure Zvar4 which was used asa reference.

TABLE 3 Tetrameric ligands, evaluated by Biacore (1 M NaOH) LigandSequence Remaining capacity 100 cycles (%) Capacity relative to ref. 100cycles Zvar4 SEQ ID NO:21 27 1 Zvar(Q9A,N11E,N28A,N43A)4 SEQ ID NO:18 552.04 Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4 SEQ ID NO:19 54 2.00Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 SEQ ID NO:20 56 2.07

Example 4

The purified tetrameric ligands of Table 2 (all with an additionalN-terminal cysteine) were immobilized on agarose beads using the methodsdescribed below and assessed for capacity and stability. The results areshown in Table 4 and FIG. 3 .

TABLE 4 Matrices with tetrametric ligands, evaluated in columns (0.5 MNaOH) Ligand SEQ ID NO. Ligand content (mg/ml) Initial IgG capacity Qb10(mg/ml) Remaining IgG capacity Qb10 after six 4 h cycles (mg/ml)Remaining IgG capacity after six 4 h cycles (%) Capacity retentionrelative to ref. after six 4 h cycles Zvar4 21 7 52.5 36.5 60 1 Zvar4 2112 61.1 43.4 71 1 Zvar(Q9A,N11E,N28A,N43A)4 18 7.0 49.1 44.1 90 1.50Zvar(Q9A,N11E,N28A,N43A)4 18 12.1 50.0 46.2 93 1.31Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 20 7.2 49.0 44.2 90 1.50Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 20 12.8 56.3 53.6 95 1.34Zvar(N11K,H18K,S33K,D37E,A42R,N43A, L44I,K50R,L51Y)4 30 9.7 56.3 52.0 921.53 Zvar(Q9A,N11K,H18K,D37E,A42R)4 31 10.8 56.9 52.5 92 1.30

Activation

The base matrix used was rigid cross-linked agarose beads of 85micrometers (volume-weighted, d50V) median diameter, prepared accordingto the methods of US6602990, hereby incorporated by reference in itsentirety, and with a pore size corresponding to an inverse gelfiltration chromatography Kav value of 0.70 for dextran of Mw 110 kDa,according to the methods described in Gel Filtration Principles andMethods, Pharmacia LKB Biotechnology 1991, pp 6-13.

25 mL (g) of drained base matrix, 10.0 mL distilled water and 2.02 gNaOH (s) was mixed in a 100 mL flask with mechanical stirring for 10 minat 25° C. 4.0 mL of epichlorohydrin was added and the reactionprogressed for 2 hours. The activated gel was washed with 10 gelsediment volumes (GV) of water.

Coupling

To 20 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 169 mgNaHCO₃, 21 mg Na₂CO₃, 175 mg NaCl and 7 mg EDTA, was added. The Falcontube was placed on a roller table for 5-10 min, and then 77 mg of DTEwas added. Reduction proceeded for >45 min. The ligand solution was thendesalted on a PD10 column packed with Sephadex G-25. The ligand contentin the desalted solution was determined by measuring the 276 nm UVabsorption.

The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH8.6} and the ligand was then coupled according to the method describedin US6399750, hereby incorporated by reference in its entirety. Allbuffers used in the experiments had been degassed by nitrogen gas for atleast 5-10 min. The ligand content of the gels could be controlled byvarying the amount and concentration of the ligand solution.

After immobilization the gels were washed 3xGV with distilled water. Thegels + 1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} wasmixed and the tubes were left in a shaking table at room temperatureovernight. The gels were then washed alternately with 3xGV {0.1 MTRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10xGV with distilledwater. Gel samples were sent to an external laboratory for amino acidanalysis and the ligand content (mg/ml gel) was calculated from thetotal amino acid content.

Protein

Gammanorm 165 mg/ml (Octapharma), diluted to 2 mg/ml in Equilibrationbuffer.

Equilibration Buffer

PBS Phosphate buffer 10 mM + 0.14 M NaCl + 0.0027 M KC1, pH 7,4(Medicago)

Adsorption Buffer

PBS Phosphate buffer 10 mM + 0.14 M NaCl + 0.0027 M KC1, pH 7,4(Medicago)

Elution Buffers

100 mM acetate pH 2.9

Dynamic Binding Capacity

2 ml of resin was packed in TRICORN™ 5 100 columns. The breakthroughcapacity was determined with an ÄKT AExplorer 10 system at a residencetime of 6 minutes (0.33 ml/min flow rate). Equilibration buffer was runthrough the bypass column until a stable baseline was obtained. This wasdone prior to auto zeroing. Sample was applied to the column until a100% UV signal was obtained. Then, equilibration buffer was appliedagain until a stable baseline was obtained.

Sample was loaded onto the column until a UV signal of 85% of maximumabsorbance was reached. The column was then washed with 5 column volumes(CV) equilibration buffer at flow rate 0.5 ml/min. The protein waseluted with 5 CV elution buffer at a flow rate of 0.5 ml/min. Then thecolumn was cleaned with 0.5 M NaOH at flow rate 0.2 ml/min andreequilibrated with equilibration buffer.

For calculation of breakthrough capacity at 10%, the equation below wasused. That is the amount of IgG that is loaded onto the column until theconcentration of IgG in the column effluent is 10% of the IgGconcentration in the feed.

$q_{10\%} = \frac{C_{0}}{V_{C}}\left\lbrack {V_{app} - V_{sys} - {\int\limits_{V_{sys}}^{V_{app}}{\frac{A(V) - A_{sub}}{A_{100\%} - A_{sub}}*dv}}} \right\rbrack$

-   A_(100%) = 100% UV signal;-   A_(sub) = absorbance contribution from non-binding IgG subclass;-   A(V) = absorbance at a given applied volume;-   V_(c) = column volume;-   V_(app) = volume applied until 10% breakthrough;-   V_(sys) = system dead volume;-   C₀ = feed concentration.

The dynamic binding capacity (DBC) at 10% breakthrough was calculated.The dynamic binding capacity (DBC) was calculated for 10 and 80%breakthrough.

CIP - 0.5 M NaOH

The 10% breakthrough DBC (Qb10) was determined both before and afterrepeated exposures to alkaline cleaning solutions. Each cycle included aCIP step with 0.5 M NaOH pumped through the column at a rate of 0.5/minfor 20 min, after which the column was left standing for 4 h. Theexposure took place at room temperature (22 +/- 2° C.). After thisincubation, the column was washed with equilibration buffer for 20 minat a flow rate of 0.5 ml/min. Table 4 shows the remaining capacity aftersix 4 h cycles (i.e. 24 h cumulative exposure time to 0.5 M NaOH), bothin absolute numbers and relative to the initial capacity.

Example 5

Example 4 was repeated with the tetrameric ligands shown in Table 5, butwith 1.0 M NaOH used in the CIP steps instead of 0.5 M. The results areshown in Table 5 and in FIG. 4 .

TABLE 5 Matrices with tetrametric ligands, evaluated in columns - 1.0 MNaOH Ligand SEQ ID NO. Ligand content (mg/ml) Initial IgG capacity Qb10(mg/ml) Remaining IgG capacity Qb10 after six 4 h cycles (mg/ml)Remaining IgG capacity after six 4 h cycles (%) Capacity retentionrelative to ref. after six 4 h cycles Zvar4 21 12 60.1 33.5 56 1Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 20 12.8 60.3 56.0 93 1.67Zvar(N11K,H18K,S33K,D37E,A42R,N43A, L44I,K50R,L51Y)4 30 9.7 62.1 48.1 771.44

Example 6 Base Matrices

The base matrices used were a set of rigid cross-linked agarose beadsamples of 59-93 micrometers (volume-weighted, d50V) median diameter(determined on a Malvern Mastersizer 2000 laser diffraction instrument),prepared according to the methods of US6602990 and with a pore sizecorresponding to an inverse gel filtration chromatography Kd value of0.62-0.82 for dextran of Mw 110 kDa, according to the methods describedabove, using HR10/30 columns (GE Healthcare) packed with the prototypesin 0.2 M NaCl and with a range of dextran fractions as probe molecules(flow rate 0.2 ml/min). The dry weight of the bead samples ranged from53 to 86 mg/ml, as determined by drying 1.0 ml drained filter cakesamples at 105° C. over night and weighing.

TABLE 6 Base matrix samples Base matrix Kd d50v (µm) Dry weight (mg/ml)A18 0.704 59.0 56.0 A20 0.70 69.2 55.8 A27 0.633 87.2 74.2 A28 0.63867.4 70.2 A29 0.655 92.6 57.5 A32 0.654 73.0 70.5 A33 0.760 73.1 55.5A38 0.657 70.9 56.2 A39 0.654 66.0 79.1 A40 0.687 64.9 74.9 A41 0.70881.7 67.0 A42 0.638 88.0 59.4 A43 0.689 87.5 77.0 A45 0.670 56.6 66.0A52 0.620 53.10 63.70 A53 0.630 52.6 86.0 A54 0.670 61.3 75.3 A55 0.64062.0 69.6 A56 0.740 61.0 56.0 A56-2 0.740 51.0 56.0 A62a 0.788 48.8 70.1A62b 0.823 50.0 46.9 A63a 0.790 66.8 59.6 A63b 0.765 54.0 79.0 A65a0.796 58.0 60.0 A65b 0.805 57.3 46.0 B5 0.793 69.0 84.4 C1 0.699 71.073.4 C2 0.642 66.5 81.1 C3 0.711 62.0 82.0 C4 0.760 62.0 82.0 H31 0.71782.0 59.0 H35 0.710 81.1 61.0 H40 0.650 52.8 65.0 I1 0.640 50.0 67.0 410.702 81.6 60.6 517 0.685 87.9 64.4 106 0.692 86.7 64.6 531C 0.661 51.763.8 P10 0.741 59.3 70.0 S9 0.736 64.1 72.2

Coupling

100 ml base matrix was washed with 10 gel volumes distilled water on aglass filter. The gel was weighed (1 g = 1 ml) and mixed with 30 mldistilled water and 8.08 g NaOH (0.202 mol) in a 250 ml flask with anagitator. The temperature was adjusted to 27 +/- 2° C. in a water bath.16 ml epichlorohydrin (0.202 mol) was added under vigorous agitation(about 250 rpm) during 90 +/- 10 minutes. The reaction was allowed tocontinue for another 80 +/- 10 minutes and the gel was then washedwith > 10 gel volumes distilled water on a glass filter until neutral pHwas reached. This activated gel was used directly for coupling as below.

To 16.4 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 139 mgNaHCO₃, 17.4 mg Na₂CO₃, 143.8 mg NaCl and 141 mg EDTA, was added. TheFalcon tube was placed on a roller table for 5-10 min, and then 63 mg ofDTE was added. Reduction proceeded for >45 min. The ligand solution wasthen desalted on a PD10 column packed with Sephadex G-25. The ligandcontent in the desalted solution was determined by measuring the 276 nmUV absorption.

The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH8.6} and the ligand was then coupled according to the method describedin US6399750 5.2.2, although with considerably higher ligand amounts(see below). All buffers used in the experiments had been degassed bynitrogen gas for at least 5-10 min. The ligand content of the gels wascontrolled by varying the amount and concentration of the ligandsolution, adding 5-20 mg ligand per ml gel. The ligand was either atetramer (SEQ ID NO:20) or a hexamer (SEQ ID NO:33) of analkali-stabilized mutant.

After immobilization the gels were washed 3xGV with distilled water. Thegels + 1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} wasmixed and the tubes were left in a shaking table at room temperatureovernight. The gels were then washed alternately with 3xGV {0.1 MTRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10xGV with distilledwater. Gel samples were sent to an external laboratory for amino acidanalysis and the ligand content (mg/ml gel) was calculated from thetotal amino acid content.

Evaluation

The Qb10 % dynamic capacity for polyclonal human IgG at 2.4 and 6 minresidence time was determined as outlined in Example 4.

TABLE 7 Prototype results Prototype Base matrix Ligand content (mg/ml)Multimer Qb10% 2.4 min (mg/ml) Qb10% 6 min (mg/ml) N1 A38 7.45 tetramer44.4 58.25 N2 A20 7.3 tetramer 45.12 57.21 N3 A42 6.72 tetramer 33.5650.02 N4 A29 7.3 tetramer 36.34 51.8 N5 A28 7.9 tetramer 42.38 58.25 N6A39 6.96 tetramer 41.88 54.67 N7 A27 7.5 tetramer 29.19 48.73 N8 A436.99 tetramer 33.43 49.79 N9 A38 11.34 tetramer 48.1 72.78 N10 A20 10.6tetramer 50.66 70.07 N11 A42 11.1 tetramer 32.25 57.78 N12 A29 11tetramer 34.85 64.68 N13 A28 11.9 tetramer 39.92 63.75 N14 A39 10.48tetramer 44.37 64.79 N15 A27 12.1 tetramer 24.8 55.56 N16 A43 10.51tetramer 31.82 58.04 N17 A41 8.83 tetramer 38.5 56.8 N18 A41 8.83tetramer 37.84 58.6 N19 A41 8.83 tetramer 35.06 57.23 N20 A41 5.0tetramer 35.64 46.04 N21 A41 13.0 tetramer 34.95 62.23 N22 A40 13.15tetramer 56.85 71.09 N23 A33 7.33 tetramer 48.69 55.76 N24 A40 11.03tetramer 54.96 73.8 033A A38 7.5 tetramer 44 58 033B A38 11.3 tetramer48 73 097A A20 7.3 tetramer 45 57 097B A20 10.6 tetramer 51 70 003A A287.9 tetramer 42 58 003B A28 11.9 tetramer 40 64 003C A28 15.8 tetramer37 67 038A A39 7.0 tetramer 42 55 038B A39 10.5 tetramer 44 65 074 A4013.2 tetramer 57 71 093 A33 7.3 tetramer 49 56 058A A40 11.0 tetramer 5574 077 A18 8.2 tetramer 52 59 010 A32 10.7 tetramer 40 57 099 A32 13.3tetramer 37 66 030A B5 6.3 tetramer 32 38 030B B5 9.6 tetramer 45 47293A C1 5.4 tetramer 38 47 293B C1 10.8 tetramer 43 60 294A C2 5.1tetramer 39 46 294B C2 10.5 tetramer 42 57 336A H40 5.6 tetramer 47 52336B H40 9.1 tetramer 52 67 091 A18 13.4 tetramer N/A 63 092 A20 12.8tetramer 49 67 080 A33 9.4 tetramer 51 58 089 A40 6.1 tetramer 49 59688A A62a 6.6 tetramer 41 46 688B A62a 14.8 tetramer 55 62 871 A62a 9.7tetramer 48 60 934A A63a 6.6 tetramer 40 44 934B A63a 14.0 tetramer 4856 017B A65a 13.1 tetramer 56 64 041A A62b 5.2 tetramer 40 N/A 041B A62b11.1 tetramer 52 N/A 116A A65b 5.8 tetramer 42 46 116B A65b 8.8 tetramer49 56 017A A65a 6.1 tetramer 40 44 387A A62a 6.4 tetramer 43 45 387BA62a 7.5 tetramer 47 56 432 A63a 6.1 tetramer 39 44 433A A65a 6.6tetramer 42 47 433B A65a 13.6 tetramer 52 61 579A I1 6.1 tetramer 45 51579B I1 11.2 tetramer 57 68 064A C3 5.9 tetramer 44 52 064B C3 9.0tetramer 49 62 064C C3 14.3 tetramer 51 70 352A C4 10.1 tetramer 55 63352B C4 14.4 tetramer 59 67 066A C3 6.8 hexamer 48 59 066B C3 11.9hexamer 51 73 066C C3 15.1 hexamer 43 61 353A C4 11.2 hexamer 62 74 353BC4 15.2 hexamer 57 82 872A A62a 9.6 hexamer 56 72 872B A62a 14.5 hexamer62 84 869A H40 6.9 hexamer 50 56 869B H40 14.3 hexamer 56 75 869C H4023.0 hexamer 41 65 962A H35 6.8 hexamer 36 49 962B H35 12.3 hexamer 3154 962C H35 20.3 hexamer 20 43 112A A56 7.9 hexamer 47 55 112B A56 12.4hexamer 57 73 112C A56 19.2 hexamer 55 80 113A A56 7.1 hexamer 48 57113B A56 12.4 hexamer 53 73 113C A56 15.2 hexamer 48 76 212A H31 6.5hexamer 37 38 212B H31 10.4 hexamer 50 61 212C H31 20.0 hexamer 31 52213A A33 6.5 hexamer 44 53 213B A33 10.9 hexamer 50 65 213C A33 11.1hexamer 50 68 432A A20 6.4 hexamer 41 56 432B A20 12.4 hexamer 38 64432C A20 21.1 hexamer 44 43 433A A38 5.9 hexamer 47 57 433B A38 11.6hexamer 48 72 433C A38 15.8 hexamer 36 62 742A A54 6.7 hexamer 38 46742B A54 12.6 hexamer 45 52 742C A54 21.1 hexamer 38 65 726A A63b 6.4hexamer 42 46 726B A63b 10.6 hexamer 49 60 726C A63b 16.7 hexamer 53 69793A A56-2 6.8 hexamer 50 58 793B A56-2 12.5 hexamer 59 72 793C A56-219.2 hexamer 61 82 517 517 12.0 tetramer* 35 56 106 106 5.8 tetramer* 3345 531C 531C 11.2 tetramer* 54 65 P10 P10 19.0 hexamer 76 S9 S9 18.4hexamer 56 75 *SEQ ID NO:21

Example 7

A series of prototypes, prepared as above, with different ligand content(tetramer, SEQ ID NO:20) were incubated in 1 M NaOH for 4, 8 and 31hours at 22 +/- 2° C. and the dynamic IgG capacity (Qb10%, 6 minresidence time) was measured before and after incubation. The prototypesare shown in Table 8 and the results in FIGS. 5 and 6 . It can be seenthat the stability towards this harsh alkali treatment increases withincreasing ligand content.

TABLE 8 Samples for incubation in 1 M NaOH Prototype Ligand content(mg/ml) Qb10%, 6 min, before incubation (mg/ml) N1 12 78 LE28 13 79 N1716 73 N16 20 73

Example 8 Pressure-Flow Testing of Matrices

300 ml sedimented matrix was packed in a FINELINE 35 column (GEHealthcare Life Sciences, Uppsala, Sweden), with 35 mm inner diameterand 330 mm tube height. The gel was suspended in distilled water toproduce a slurry volume of 620 ml and the height of the packed bed was300 +/- 10 mm. The packing pressure was 0.10 +/- 0.02 bar (10 +/- 2kPa).

Distilled water was then pumped through the column at increasing pumprates and the flow rate (expressed as the linear flow velocity, cm/h)and back pressure (MPa) was measured after 5 min for each pump setting.The measurements were continued until a max flow rate and a max pressurewas reached, i.e. the flow rate and back pressure achieved when the flowrate starts to diminish at increasing back pressures.

TABLE 9 Pressure flow performance of matrices Matrix Max flow velocity,cm/h Max pressure (MPa) 517 1343 0.56 106 1306 0.56 531C 513 0.51 P10862 0.60 S9 1172 0.64

The P10 and S9 matrices have a higher rigidity, as indicated by the maxpressure, and can thus sustain comparatively high flow velocitiesdespite their low (59-64 micrometers) median particle diameters.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims. All patents and patentapplications mentioned in the text are hereby incorporated by referencein their entireties, as if they were individually incorporated.

ITEMIZED LIST OF EMBODIMENTS

i. An Fc-binding polypeptide which comprises a sequence as defined by,or having at least 90% or at least 95% or 98% identity to SEQ ID NO:53.

X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉ SX₁₀X₁₁X₁₂LAEAKX₁₃X₁₄NX₁₅AQ (SEQ ID NO:53)

wherein individually of each other:

-   X₁=A or Q or is deleted-   X₂=E,K,Y,T,F,L,W,I,M,V,A,H or R-   X₃=H or K-   X₄=A or N-   X₅=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K-   X₆=Q or E-   X₇=S or K-   X₈=E or D-   X₉=Q or V or is deleted-   X₁₀=K,R or A or is deleted-   X₁₁=A,E or N or is deleted-   X₁₂=I or L-   X₁₃=K or R-   X₁₄=L or Y-   X₁₅=D, F,Y,W,K or R

ii. The polypeptide of embodiment i, wherein:

X₁ = A or is deleted, X₂ = E, X₃ = H, X₄ = N, X₆ = Q, X₇ = S, X₈ = D, X₉= V or is deleted, X₁₀ = K or is deleted, X₁₁ = A or is deleted, X₁₂ =I, X₁₃ = K, X₁₄ = L.

iii. The polypeptide of embodiment i or ii, wherein X₁=A, X₂ = E, X₃ =H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀ = K, X₁₁ = A, X₁₂ =I, X₁₃ = K, X₁₄ = L and X₁₅=D.

iv. The polypeptide of embodiment i or ii, wherein X₁ is deleted, X₂ =E, X₃ = H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀ = K, X₁₁ =A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

v. The polypeptide of embodiment i or ii, wherein X₁=A, X₂ = E, X₃ = H,X₄ = N, Xs= S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X₆ = Q, X₇ = S, X₈ = D,X₉ = V, X₁₀ = K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

vi. The polypeptide of embodiment i or ii, wherein X₁=A, X₂ = E, X₃ = H,X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ is deleted, X₁₀ = K, X₁₁ = A,X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

vii. The polypeptide of embodiment i or ii, wherein X₁=A, X₂ = E, X₃ =H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀ is deleted, X₁₁ =A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

viii. The polypeptide of embodiment i or ii, wherein X₁=A, X₂ = E, X₃ =H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀ = K, X₁₁ isdeleted, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

ix. The polypeptide of embodiment i or ii, wherein X₁=A, X₂ = E, X₃ = H,X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀ = K, X₁₁ = A, X₁₂ = I,X₁₃ = K, X₁₄ = L and X₁₅= F,Y,W,K or R.

x. An Fc-binding polypeptide comprising a mutant of a parentalFc-binding domain of Staphylococcus Protein A (SpA), as defined by, orhaving at least 90% such as at least 95% or 98% identity to, SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO:51 or SEQ ID NO:52, wherein atleast the asparagine or serine residue at the position corresponding toposition 11 in SEQ ID NO:4-7 has been mutated to an amino acid selectedfrom the group consisting of glutamic acid, lysine, tyrosine, threonine,phenylalanine, leucine, isoleucine, tryptophan, methionine, valine,alanine, histidine and arginine.

xi. The polypeptide of embodiment x, comprising a mutant of a parentalFc-binding domain of Staphylococcus Protein A (SpA), as defined by, orhaving at least 90% such as at least 95% or 98% identity to, SEQ IDNO:51 or SEQ ID NO:52.

xii. The polypeptide of embodiment x or xi, wherein the amino acidresidue at the position corresponding to position 11 in SEQ ID NO:4-7 isa glutamic acid.

xiii. The polypeptide of any one of embodiments x-xii, wherein the aminoacid residue at the position corresponding to position 11 in SEQ IDNO:4-7 is a lysine.

xiv. The polypeptide of any one of embodiments x-xiii, wherein the aminoacid residue at the position corresponding to position 29 in SEQ IDNO:4-7 is a glycine, serine, tyrosine, glutamine, threonine, asparagine,phenylalanine, leucine, tryptophan, isoleucine, methionine, valine,aspartic acid, glutamic acid, histidine, arginine or lysine.

xv. The polypeptide of any one of embodiments x-xiv, wherein the aminoacid residue at the position corresponding to position 9 in SEQ IDNO:4-7 is an alanine.

xvi. The polypeptide of any one of embodiments x-xv, wherein the aminoacid residue at the position corresponding to position 9 in SEQ IDNO:4-7 has been deleted.

xvii. The polypeptide of any one of embodiments x-xvi, wherein the aminoacid residue at the position corresponding to position 50 in SEQ IDNO:4-7 is an arginine or a glutamic acid, such as an arginine.

xviii. The polypeptide of any one of embodiments x-xvii, wherein theamino acid residue at the position corresponding to position 43 in SEQID NO:4-7 has been deleted.

xix. The polypeptide of any one of embodiments x-xviii, wherein theamino acid residue at the position corresponding to position 28 in SEQID NO:4-7 is an alanine or an asparagine.

xx. The polypeptide of any one of embodiments x-xix, wherein the aminoacid residue at the position corresponding to position 40 in SEQ IDNO:4-7 is selected from the group consisting of asparagine, alanine,glutamic acid and valine.

xxi. The polypeptide of any one of embodiments x-xx, wherein the aminoacid residue at the position corresponding to position 40 in SEQ IDNO:4-7 has been deleted.

xxii. The polypeptide according to any one of embodiments x-xxi, whereinthe amino acid residue at the position corresponding to position 42 inSEQ ID NO:4-7 is an alanine, lysine or arginine, such as an arginine.

xxiii. The polypeptide according to any one of embodiments x-xxii,wherein the amino acid residue at the position corresponding to position42 in SEQ ID NO:4-7 has been deleted.

xxiv. The polypeptide according to any one of embodiments x-xxiii,wherein the amino acid residue at the position corresponding to position44 in SEQ ID NO:4-7 is a leucine or an isoleucine, such as anisoleucine.

xxv. The polypeptide according to any one of embodiments x-xxiv, whereinthe amino acid residue at the position corresponding to position 44 inSEQ ID NO:4-7 has been deleted.

xxvi. The polypeptide according to any one of embodiments x-xxv, whereinthe amino acid residue at the position corresponding to position 53 inSEQ ID NO:4-7 is a phenylalanine, a tyrosine, a tryptophan, an arginineor a lysine.

xxvii. The polypeptide according to any one of embodiments x-xxvi,wherein the amino acid residue at the position corresponding to position18 in SEQ ID NO:4-7 is a lysine or a histidine, such as a lysine.

xxviii. The polypeptide according to any one of embodiments x-xxvii,wherein the amino acid residue at the position corresponding to position33 in SEQ ID NO:4-7 is a lysine or a serine, such as a lysine.

xxix. The polypeptide according to any one of embodiments x-xxviii,wherein the amino acid residue at the position corresponding to position37 in SEQ ID NO:4-7 is a glutamic acid or an aspartic acid, such as aglutamic acid.

xxx. The polypeptide according to any one of embodiments x-xxix, whereinthe amino acid residue at the position corresponding to position 51 inSEQ ID NO:4-7 is a tyrosine or a leucine, such as a tyrosine.

xxxi. The polypeptide according to any one of embodiments x-xxx, whereinone or more of the amino acid residues at the positions corresponding topositions 1, 2, 3, 4, 5, 6, 7, 8, 56, 57 or 58 in SEQ ID NO: 4-7 havebeen deleted.

xxxii. The polypeptide according to any one of embodiments x-xxxi,wherein the mutation is selected from the group consisting of:

-   Q9A,N11E, A29G,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29S,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29Y,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29Q,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29T,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29N,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29F,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29L,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29W,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29I,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29M,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29V,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29D,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29E,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29H,Q40V,A42K,N43A,L44I;-   Q9A,N11E, A29R,Q40V,A42K,N43A,L44I; and-   Q9A,N11E, A29K,Q40V,A42K,N43A,L44I.

xxxiii. The polypeptide according to any one of embodiments x-xxxii,wherein the mutation is selected from the group consisting of:

-   Q9A,N11E, Q40V,A42K,N43A,L44I,D53F;-   Q9A,N11E, Q40V,A42K,N43A,L44I,D53Y;-   Q9A,N11E, Q40V,A42K,N43A,L44I,D53W;-   Q9A,N11E, Q40V,A42K,N43A,L44I,D53K; and-   Q9A,N11E, Q40V,A42K,N43A,L44I,D53R.

xxxiv. The polypeptide according to any one of embodiments x-xxxiii,wherein the mutation is selected from the group consisting of:

-   Q9de1,N11E, Q40V,A42K,N43A,L44I;-   Q9A,N11E, Q40del,A42K,N43A,L44I;-   Q9A,N11E, Q40V,A42de1,N43A,L44I; and-   Q9A,N11E, Q40V,A42K,N43del,L44I.

xxxv. The polypeptide according to any one of embodiments x-xxxiv,wherein the mutation is selected from the group consisting of:

-   D2de1,A3de1,K4de1,Q9A,N11E,Q40V,A42K,N43A,L44I;-   V1de1,D2de1,Q9A,N11E,Q40V,A42K,N43A,L44I,K58de1;-   V1de1,D2de1,A3de1,Q9A,N11E,Q40V,A42K,N43A,L44I,P57de1,K58de1;-   K4de1,F5de1,D6de1,K7de1,E8de1,Q9A,N11E,Q40V,A42K,N43A,L44I;-   Q9A,N11E,Q40V,A42K,N43A,L44I,A56de1,P57de1,K58de1;-   V1de1,,D2de1,A3de1,Q9A,N11E,Q40V,A42K,N43A,L44I;-   V1de1,D2de1,A3de1,K4de1,F5de1,D6de1,K7    de1,E8de1,Q9A,N11E,Q40V,A42K,N43A,L44I;and-   Q9A,N11E,Q40V,A42K,N43A,L44I,K58_insYEDG.

xxxvi. The polypeptide according to any one of embodiments i-xxxi,comprising or consisting essentially of a sequence having at least 90%identity to an amino acid sequence selected from the group consistingof: SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69 and SEQ ID NO:70.

xxxvii. The polypeptide according to any one of embodiments i-xxxi,comprising or consisting essentially of a sequence having at least 90%identity to an amino acid sequence selected from the group consistingof: SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ IDNO:75.

xxxviii. The polypeptide according to any one of embodiments i-xxxi,comprising or consisting essentially of a sequence having at least 90%identity to an amino acid sequence selected from the group consistingof: SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79.

xxxix. The polypeptide according to any one of embodiments i-xxxi,comprising or consisting essentially of a sequence having at least 90%identity to an amino acid sequence selected from the group consistingof: SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ IDNO:93, SEQ ID NO:94 and SEQ ID NO:95.

xl. The polypeptide according to any preceding embodiment, whichpolypeptide has an improved alkaline stability compared to a polypeptideas defined by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:7, such as by SEQ ID NO:7.

xli. The polypeptide according to any preceding embodiment, whichpolypeptide has an improved alkaline stability compared to a parentalpolypeptide as defined by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, such as by SEQ ID NO:7.

xlii. The polypeptide according to embodiment xl or xli, wherein thealkaline stability is improved as measured by the remaining IgG-bindingcapacity, after 24, 25 h incubation in 0.5 M or 1.0 M aqueous NaOH at 22+/- 2° C.

xliii. A multimer comprising or consisting essentially of a plurality ofpolypeptides as defined by any preceding embodiment.

xliv. The multimer according to embodiment xliii, wherein thepolypeptides are linked by linkers comprising up to 25 amino acids, suchas 3-25 or 3-20 amino acids.

xlv. The multimer of embodiment xliii or xliv, wherein at least twopolypeptides are linked by linkers comprising or consisting essentiallyof a sequence having at least 90% identity with an amino acid sequenceselected from the group consisting of APKVDAKFDKE (SEQ ID NO:96),APKVDNKFNKE (SEQ ID NO:97), APKADNKFNKE (SEQ ID NO:98), APKVFDKE (SEQ IDNO:99), APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQ ID NO:101), APKVDA (SEQID NO: 102), VDAKFDKE (SEQ ID NO:103), APKKFDKE (SEQ ID NO: 104), APK,APKYEDGVDAKFDKE (SEQ ID NO: 105) and YEDG (SEQ ID NO:106).

xlvi. The multimer according to embodiment xliv or xlv, which is adimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer ornonamer.

xlvii. The multimer according to any one of embodiments xliv-xlvi, whichcomprises or consists essentially of a sequence selected from the groupof sequences defined by SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87.

xlviii. The polypeptide or multimer according to any precedingembodiment, further comprising at, or within 1-5 amino acid residuesfrom, the C-terminal or N-terminal one or more coupling element,selected from the group consisting of one or more cysteine residues, aplurality of lysine residues and a plurality of histidine residues.

xlix. A nucleic acid or a vector encoding a polypeptide or multimeraccording to any preceding embodiment.

1. An expression system, which comprises a nucleic acid or vectoraccording to embodiment xlix.

li. A separation matrix, wherein a plurality of polypeptides ormultimers according to any one of embodiment i - xlviii have beencoupled to a solid support.

lii. A separation matrix comprising at least 11 mg/ml Fc-binding ligandscovalently coupled to a porous support, wherein:

-   a) said ligands comprise multimers of alkali-stabilized Protein A    domains,-   b) said porous support comprises cross-linked polymer particles    having a volume-weighted median diameter (d50,v) of 55-70    micrometers and a dry solids weight of 55-80 mg/ml.

liii. A separation matrix comprising at least 15, such as 15-21 or 15-18mg/ml Fc-binding ligands covalently coupled to a porous support, whereinsaid ligands comprise multimers of alkali-stabilized Protein A domains.

liv. The separation matrix of embodiment li or liii, wherein saidcross-linked polymer particles comprise cross-linked polysaccharideparticles.

lv. The separation matrix of any one of embodiments li-liv, wherein saidcross-linked polymer particles comprise cross-linked agarose particles.

lvi. The separation matrix of any one of embodiments li-lv, wherein saidcross-linked polymer particles have a pore size corresponding to aninverse gel filtration chromatography Kd value of 0.70-0.85 for dextranof Mw 110 kDa.

lvii. The separation matrix of any one of embodiments li-lvi, which hasa max pressure of at least 0.58, such as at least 0.60, MPa when packedat 300 +/-10 mm bed height in a FINELINE 35 column.

lviii. The separation matrix of any one of embodiments li-lvii, whereinsaid multimers comprise tetramers, pentamers, hexamers or heptamers ofalkali-stabilized Protein A domains.

lix. The separation matrix of any one of embodiments li-lviii, whereinsaid multimers comprise hexamers of alkali-stabilized Protein A domains.

lx. The separation matrix of any one of embodiments li-lix, wherein thepolypeptides are linked by linkers comprising up to 25 amino acids, suchas 3-25 or 3-20 amino acids.

lxi. The separation matrix of any one of embodiments li-lx, wherein atleast two polypeptides are linked by linkers comprising or consistingessentially of a sequence having at least 90% identity with an aminoacid sequence selected from the group consisting of APKVDAKFDKE (SEQ IDNO:96), APKVDNKFNKE (SEQ ID NO:97), APKADNKFNKE (SEQ ID NO:98), APKVFDKE(SEQ ID NO:99), APAKFDKE (SEQ ID NO:100), AKFDKE (SEQ ID NO:101), APKVDA(SEQ ID NO:102), VDAKFDKE (SEQ ID NO:103), APKKFDKE (SEQ ID NO:104),APK, APKYEDGVDAKFDKE (SEQ ID NO:105) and YEDG (SEQ ID NO:106).

lxii. The separation matrix of any one of embodiments li-lxi, having a10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml,such as at least 50 or at least 55 mg/ml mg/ml at 2.4 min residencetime.

lxiii. The separation matrix of any one of embodiments li-lxii, having a10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml,such as at least 65, at least 70 or at least 75 mg/ml at 6 min residencetime.

lxiv. The separation matrix of any one of embodiments li-lxiii, whereinthe 10% breakthrough dynamic binding capacity for IgG at 2.4 or 6 minresidence time is reduced by less than 20 % after incubation 31 h in 1.0M aqueous NaOH at 22 +/- 2 C.

lxv. The separation matrix of any one of embodiments li-lxiv, having adissociation constant for IgG2 of below 0.2 mg/ml, such as below 0.1mg/ml, in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.

lxvi. The separation matrix according to any one of embodiments li-lxv,wherein the polypeptides or multimers have been coupled to the solidsupport or porous support via thioether bonds.

lxvii. The separation matrix according to any one of embodiments li -lxvi, wherein the solid support or porous support is a polysaccharide.

lxviii. The separation matrix according to any one of embodimentsli-lxvii, wherein the IgG capacity of the matrix after 24 incubation in0.5 M NaOH at 22 +/- 2° C. is at least 80, such as at least 85, at least90 or at least 95% of the IgG capacity before the incubation.

lxix. The separation matrix according to any one of embodimentsli-lxviii, wherein the IgG capacity of the matrix after 24 incubation in1.0 M NaOH at 22 +/- 2° C. is at least 70, such as at least 80 or atleast 90% of the IgG capacity before the incubation.

lxx. The separation matrix of any one of embodiments li-lxix, whereinsaid alkali-stabilized Protein A domains or plurality ofpolypeptides/multimers comprise(s) mutants of a parental Fc-bindingdomain of Staphylococcus Protein A (SpA), as defined by, or having atleast 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:22, SEQ ID NO:51 or SEQ ID NO:52, wherein at leastthe asparagine or serine residue at the position corresponding toposition 11 in SEQ ID NO:4-7 has been mutated to an amino acid selectedfrom the group consisting of glutamic acid, lysine, tyrosine, threonine,phenylalanine, leucine, isoleucine, tryptophan, methionine, valine,alanine, histidine and arginine.

lxxi. The separation matrix of embodiment lxx, wherein the amino acidresidue at the position corresponding to position 11 in SEQ ID NO:4-7is, or has been mutated to, a glutamic acid or a lysine.

lxxii. The separation matrix of embodiment lxx or lxxi, wherein theamino acid residue at the position corresponding to position 40 in SEQID NO:4-7 is, or has been mutated to, a valine.

lxxiii. The separation matrix of any one of embodiments li-lxix, whereinsaid alkali-stabilized Protein A domains or plurality ofpolypeptides/multimers comprise(s) an Fc-binding polypeptide having anamino acid sequence as defined by, or having at least 80%, such as atleast 90, 95 or 98%, identity to SEQ ID NO:53.

X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉ SX₁₀X₁₁X₁₂LAEAKX₁₃X₁₄NX₁₅AQ (SEQ ID NO:53)

wherein individually of each other:

-   X₁=A or Q or is deleted-   X₂=E,K,Y,T,F,L,W,I,M,V,A,H or R-   X₃=H or K-   X₄=A or N-   X₅=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K-   X₆=Q or E-   X₇=S or K-   X₈=E or D-   X₉=Q or V or is deleted-   X₁₀=K,R or A or is deleted-   X₁₁=A,E or N or is deleted-   X₁₂=I or L-   X₁₃=K or R-   X₁₄=L or Y-   X₁₅=D, F,Y,W,K or R

lxxiv. The separation matrix of embodiment lxxiiii, wherein individuallyof each other:

X₁ = A or is deleted, X₂ = E, X₃ = H, X₄ = N, X₆ = Q, X₇ = S, X₈ = D, X₉= V or is deleted, X₁₀ = K or is deleted, X₁₁ = A or is deleted, X₁₂ =I, X₁₃ = K, X₁₄ = L.

lxxv. The separation matrix of embodiment lxxiii, wherein individuallyof each other:

X₁=A, X₂ = E, X₃ = H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V, X₁₀= K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

lxxvi. The separation matrix of embodiment lxxiii, wherein individuallyof each other:

wherein X₁ is A, X₂ = E, X₃ = H, X₄ = N, X₆ = Q, X₇ = S, X₈ = D, X₉ = V,X₁₀ = K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

lxxvii. The separation matrix of embodiment lxxiii, wherein individuallyof each other:

wherein X₁ is A, X₃ = H, X₄ = N, X₅=A, X₆ = Q, X₇ = S, X₈ = D, X₉ = V,X₁₀ = K, X₁₁ = A, X₁₂ = I, X₁₃ = K, X₁₄ = L and X₁₅=D.

lxxviii. The separation matrix according to any one of embodimentsli-lxxvii, wherein said multimers or polypeptides further comprise at,or within 1-5 amino acid residues from, the C-terminal or N-terminal oneor more coupling element, selected from the group consisting of one ormore cysteine residues, a plurality of lysine residues and a pluralityof histidine residues.

lxxix. The separation matrix according to any one of embodimentsli-lxxviii wherein said multimers or polypeptides further comprise atthe N-terminal a leader sequence, comprising 1-20 amino acid residues.

lxxx. A method of isolating an immunoglobulin, wherein a separationmatrix according to any one of embodiments li-lxxix is used.

lxxxi. The method of embodiment lxxx, comprising the steps of:

-   a) contacting a liquid sample comprising an immunoglobulin with a    separation matrix according to any one of embodiments li-lxxix,-   b) washing said separation matrix with a washing liquid,-   c) eluting the immunoglobulin from the separation matrix with an    elution liquid, and-   d) cleaning the separation matrix with a cleaning liquid.

lxxxii. The method of embodiment lxxxi, wherein the cleaning liquid isalkaline, such as with a pH of 13-14.

lxxxiii. The method of embodiment lxxxi or lxxxii, wherein the cleaningliquid comprises 0.1 -1.0 M NaOH or KOH, such as 0.5 - 1.0 M or 0.4-1.0M NaOH or KOH.

lxxxiv. The method of any one of embodiments lxxxi - lxxxiii, whereinsteps a) - d) are repeated at least 10 times, such as at least 50 timesor 50 - 200 times.

lxxxv. The method of any one of embodiments lxxxi -lxxxiv, wherein stepsa) - c) are repeated at least 10 times, such as at least 50 times or50 - 200 times and wherein step d) is performed after a plurality ofinstances of step c), such as at least 10 or at least 50 times.

1. A separation matrix comprising at least 15 mg/ml Fc-binding ligandscovalently coupled to a porous support, wherein: the ligands comprisemultimers of alkali-stabilized Protein A domains; the porous supportcomprises cross-linked polymer particles having a volume-weighted mediandiameter (d50,v) of from 56 µm to 70 µm; the separation matrix has a maxpressure of at least 0.58 MPa when packed at 300 +/- 10 mm bed height ina 35 mm separation column; and the Protein A domains comprise anFc-binding polypeptide defined by, or having at least 80% identity toSEQ ID NO: 53 and X₁Q X₂AFYEILX₃LP NLTEEQRX₄X₅F IX₆X₇LKDX₈PSX₉SX₁₀X₁₁X₁₂LAEAKX₁₃ X₁₄NX₁₅AQ (SEQ ID NO: 53), wherein individually ofeach other: X₁ = A or Q or is deleted; X₂ = E, K, Y, T, F, L, W, I, M,V, A, H, or R; X₃ = H or K; X₄ = A or N; X₅ = A, G, S, Y, Q, T, N, F, L,W, I, M, V, D, E, H, R, or K; X₆ = Q or E; X₇ = S or K; X₈ = E or D; X₉= W or V or is deleted; X₁₀ = K, R, or A, or is deleted; X₁₁ = A, E, orN, or is deleted; X₁₂ = I or L; X₁₃ = K or R; X₁₄ = L or Y; and X₁₅ = D,F, Y, W, K, or R.
 2. The separation matrix of claim 1, wherein thecross-linked polymer particles have a dry solids weight of from 55 mg/mlto 80 mg/ml.
 3. The separation matrix of claim 1, wherein thecross-linked polymer particles have a dry solids weight of from 60 mg/mlto 78 mg/ml.
 4. The separation matrix of claim 1, wherein thecross-linked polymer particles have a dry solids weight of from 65 mg/mlto 78 mg/ml.
 5. The separation matrix of claim 1, wherein thecross-linked polymer particles have a volume-weighted median diameter(d50,v) of from 56 µm to 66 µm.
 6. The separation matrix of claim 1,wherein the cross-linked polymer particles comprise cross-linkedpolysaccharide particles.
 7. The separation matrix of claim 1, whereinthe cross-linked polymer particles comprise cross-linked agaroseparticles.
 8. The separation matrix of claim 1, wherein the cross-linkedpolymer particles have a pore size corresponding to an inverse gelfiltration chromatography Kd value of 0.69-0.85 for dextran of Mx 100kDa.
 9. The separation matrix of claim 1, wherein the multimers comprisetetramers, pentamers, hexamers, or heptamers of alkali-stabilizedProtein A domains.
 10. The separation matrix of claim 1, wherein themultimers comprise hexamers of alkali-stabilized Protein A domains. 11.The separation matrix of claim 1, having a 10% breakthrough dynamicbinding capacity for IgG of at least 45 mg/ml at 2.4 minutes residencetime.
 12. The separation matrix of claim 1, having a 10% breakthroughdynamic binding capacity for IgG of at least 50 mg/ml at 2.4 minutesresidence time.
 13. The separation matrix of claim 1, having a 10%breakthrough dynamic binding capacity for IgG of at least 55 mg/ml at2.4 minutes residence time.
 14. The separation matrix of claim 1, havinga 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/mlat 6 minutes residence time.
 15. The separation matrix of claim 1,having a 10% breakthrough dynamic binding capacity for IgG of at least65 mg/ml at 6 minutes residence time.
 16. The separation matrix of claim1, having a 10% breakthrough dynamic binding capacity for IgG of atleast 70 mg/ml at 6 minutes residence time.
 17. The separation matrix ofclaim 1, having a 10% breakthrough dynamic binding capacity for IgG ofat least 75 mg/ml at 6 minutes residence time.
 18. The separation matrixof claim 11, wherein the 10% breakthrough dynamic binding capacity forIgG at 2.4 minutes residence time is reduced by less than 20% afterincubation for 31 hours in 1.0 M aqueous NaOH at 22 +/- 2° C.
 19. Theseparation matrix of claim 1, having a dissociation constant for IgG2 ofbelow 0.2 mg/ml in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.
 20. Theseparation matrix of claim 1, having a dissociation constant for IgG2 ofbelow 0.1 mg/ml in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.
 21. Theseparation matrix of claim 1, wherein individually of each other: X₁ = Aor is deleted; X₂ = E; X₃ = H; X₄ = N; X₆ = Q; X₇ = S; X₈ = D; X₉ = V oris deleted; X₁₀ = K or is deleted; X₁₁ = A or is deleted; X₁₂ = I; X₁₃ =K; and X₁₄ = L.
 22. The separation matrix of claim 1, wherein the aminoacid sequence has at least 90% identity to SEQ ID NO:
 53. 23. Theseparation matrix of claim 1, wherein the amino acid sequence has atleast 95% identity to SEQ ID NO:
 53. 24. The separation matrix of claim1, wherein the amino acid sequence has at least a 98% identity to SEQ IDNO:
 53. 25. The separation matrix of claim 1, wherein the amino acidsequence is identical to SEQ ID NO:
 53. 26. The separation matrix ofclaim 1, wherein the polypeptides are linked by linkers comprising up to25 amino acids.
 27. The separation matrix of claim 1, wherein at leasttwo polypeptides are linked by linkers comprising or consistingessentially of a sequence having at least 90% identity with an aminoacid sequence selected from the group consisting of APKVDAKFDKE (SEQ IDNO: 96), APKVDNKFNKE (SEQ ID NO: 97), APKADNKFNKE (SEQ ID NO: 98),APKVFDKE (SEQ ID NO: 99), APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQ ID NO:101), APKVDA (SEQ ID NO: 102), VDAKFDKE (SEQ ID NO: 103), APKKFDKE (SEQID NO: 104), APK, APKYEDGVDAKFDKE (SEQ ID NO: 105), and YEDG (SEQ ID NO:106).
 28. The separation matrix of claim 1, comprising from 15 mg/ml to21 mg/ml Fc-binding ligands covalently coupled to the porous support.29. The separation matrix of claim 1, comprising from 17 mg/ml to 21mg/ml Fc-binding ligands covalently coupled to the porous support. 30.The separation matrix of claim 1, comprising from 18 mg/ml to 20 mg/mlFc-binding ligands covalently coupled to the porous support.
 31. Amethod of isolating an immunoglobulin, comprising the steps of: a)contacting an immunoglobulin with a separation matrix according to claim1; b) washing the separation matrix with a washing liquid; c) elutingthe immunoglobulin from the separation matrix with an elution liquid;and d) cleaning the separation matrix with a cleaning liquid.
 32. Themethod of claim 32, wherein the cleaning liquid comprises from 0.1 to1.0 M NaOH or KOH.
 33. The method of claim 32, wherein steps a) - d) arerepeated at least 10 times.
 34. The method of claim 32, wherein in stepd) the elution liquid has a pH of from 2.5 to 4.5.
 35. The method ofclaim 32, wherein in step b) the pH is from 6 to
 8. 36. The method ofclaim 32, wherein in step b) the residence time of the liquid on theseparation matrix is from 2 minutes to 20 minutes.
 37. The separationmatrix of claim 32, wherein the cleaning liquid comprises at least 0.5 MNaOH.
 38. The separation matrix of claim 33, wherein in step e) thecontact time between the separation matrix and the cleaning liquid isless than 10 minutes.